Xanthomonas Genomics Conference 2015 - xgc 2015 - Universidad [PDF]

2015

Xanthomonas Genomics Conference July 8th - 11th, 2015 Bogotá - Colombia

This book of abstracts was printed thanks to the support of UNU-BIOLAC

Organized by: Universidad de los Andes CORPOICA, Corporación Colombiana para la Investigación Agropecuaria Insitut de Recherche pour le Développement – IRD, Montpellier, France Universidad Nacional de Colombia Supported by: Universidad de los Andes UNU-BIOLAC – United Nations University - Biotechnology Programme for Latin America and the Caribbean CORPOICA – Corporación Colombiana de Investigación Agropecuaria Embassy of France in Colombia and Institut Français - Colombia CIAT - International Center for Tropical Agriculture ICA – Instituto Colombiano Agropecuario FPIT - Fundación para la Promoción de la Investigación y la Tecnología – Banco de la República de Colombia

2015

Xanthomonas Genomics Conference

BOOK OF ABSTRACTS 5th Xanthomonas Genomics Conference July 8th – 11th, 2015 Bogotá, Colombia

SCIENTIFIC COMMITTEE Adriana Bernal (Universidad de los Andes. Bogotá, Colombia) Ralf Koebnik (Insitut de Recherche pour le Développement. Montpellier, France) Carolina Gonzalez (Corporacion Colombiana de Investigación Agropecuaria – CORPOICA. Colombia) Camilo López (Universidad Nacional de Colombia. Bogotá, Colombia) Jens Boch (Martin Luther Universität. Halle-Wittenberg, Germany) Pamela Ronald (University of California, Davis. California, USA) Marie Agnes Jacques (Institut National de la Recherche Agronomique. Angers, France) Gongyou Chen (Shanghai Jiao Tong University. Shanghai, China)

ORGANIZING COMMITTEE Adriana Bernal (Universidad de los Andes. Bogotá, Colombia) Vivian Bernal (Universidad de los Andes. Bogotá, Colombia) Ralf Koebnik (Insitut de Recherche pour le Développement – IRD, Montpellier, France) Carolina Gonzalez (Corporacion Colombiana de Investigación Agropecuaria – CORPOICA, Colombia) Camilo López (Universidad Nacional de Colombia. Bogotá, Colombia) Boris Szurek (Insitut de Recherche pour le Développement – IRD, Montpellier, France) Valerie Verdier (Insitut de Recherche pour le Développement – IRD, Montpellier, France)

SCIENTIFIC PROGRAM

Wednesday

July 8th, 2015

14:00

Hotel – University Transportation

14:30

Registration

16:00

Welcoming words

Opening lecture 16:30

Dean Gabriel (Opening lecture) Blights, Cankers, Spots, Toxins and Dissemination of Xanthomonas

17:10

Cultural show and welcoming Cocktail

19:10

University - Hotel Transportation

Thursday

July 9th, 2015

6:00

Breakfast at the hotel

8:00

Hotel - University Transportation

Session I: Diversity and Diagnosis Chairwoman: Carolina González 8:30

Olivier Pruvost (Invited speaker) Tandem Repeats as Genotyping Tools for Understanding the Genetic Structure and Epidemiology of Xanthomonas citri pv. citri

9:10

Taca Vancheva. Genotyping of Xanthomonas euvesicatoria Strains Using Multiple- Locus Variable Number Tandem Repeat Analysis

9:30

Bart Cottyn. The Diversity of Xanthomonas axonopodis pv. dieffenbachiae Demands a Taxonomic Revision for Accurate Diagnosis

9:50

Coffee Break + Poster viewing

10:20

Michael Gétaz. Improving Detection of Strawberry Plant Material Infected by the Quarantine Bacterium Xanthomonas fragariae: Development of a LAMP Assay as Diagnostic Tool for On-Field Specific Identification

10:40

Valérie Verdier. The Power of Genomics in Xanthomonas Diagnostics and Taxonomy

Session II: Epidemiology and Ecology Chairman: Lionel Gagnevin 11:00

Jan Leach (Invited speaker) Coping With Combined Stresses: Rice, Global Warming, And Bacterial Disease

11:40

Déborah Merda. Xanthomonas arboricola Fits the Epidemic Bacterial Population Model with Epidemic Clones Superimposed Upon a Recombinant Network

12:00

Marion Fischer-Le Saux. Evolution and Comparative Genomics of Pathogenic and Nonpathogenic Strains of Xanthomonas arboricola Unveil Evolutionary Mechanisms of Pathoadaptation

12:20

Lunch

Session III: Pathogenesis and Regulation Chairman: Gongyou Chen

13:50

Nian Wang (Invited speaker) Exploration of the Post-Transcriptional Regulation of T3SS Genes in Xanthomonas citri

14:30

Ralf Koebnik. Yes We Can – Cannabis as a Promising Host to Study Pathogenicity of Xanthomonads

14:50

Lena Hersemann. Pathogenomics of Xanthomonas translucens pv. graminis to Unravel its Virulence on Forage Grasses

15:10

Cornelius Schmidtke. RNA-Mediated Regulation of Virulence of Xanthomonas campestris pv. vesicatoria

15:30

Coffee break + Poster viewing

Poster session 15:50

Poster exhibition

17:40

University - Hotel Transportation

Friday

July 10th, 2015

6:00

Breakfast at the hotel

8:00

Hotel-University Transportation

Session III: Pathogenesis and Regulation (continued) Chairman: Gongyou Chen 8:30

Mathieu Arlat (Invited speaker) CUT Systems of Xanthomonas and PUL Systems of Bacteroidetes Gut Symbionts: A Convergent Evolution to Exploit Plant Compounds?

9:00

Marie Agnès Jacques. A Single Chemotaxis Sensor Defines Xanthomonas campestris Host Range and Restrict it to Brassicaceae.

9:20

Chenyang He. A Novel Cyclic di-GMP Signalling Pathway Mediated by the PdeRTrip Interaction in Regulation of Virulence of Xanthomonas oryzae pv. oryzae

9:40

Coffee Break

Session IV: Xop Effector Biology Chairman: Ralf Koebnik 10:10

Ulla Bonas (Invited speaker) Type III Effectors of Xanthomonas campestris pv. vesicatoria

10:50

Suayib Üstün. Interplay Between SA Signaling and Xanthomonas Type III Effector XopJ During its Virulence and Avirulence Function

11:10

César Medina. Characterization of Type Three Effectors from Xanthomonas axonopodis pv. manihotis in Virulence and Suppression of Plant Innate Immunity

11:30

Rebecca Bart. The Role of the Environment in Host Specificity, Pathogen Virulence and Disease Spread

11:50

Guido Sessa. Identification and Functional Analysis of Novel Xanthomonas euvesicatoria Type III Effectors

12:10

Lunch

Session V: TAL Effector Biology Chairman: Boris Szurek 13:40

Adam J. Bogdanove (Invited speaker) Moderately High-Throughput Xanthomonas Population-Level TAL Effector Analysis

Genome

Sequencing

for

14:20

Alvaro Pérez-Quintero. QueTAL: A Suite of Tools to Classify and Compare TAL Effectors Functionally and Phylogenetically

14:40

Lionel Gagnevin. Which Effectors in the TAL Arsenal of Xanthomonas citri pv. citri are Responsible for Host Specificity on Citrus?

15:00

Gong-You Chen. Transcriptional Activator-Like Effectors TAL6 and TAL11a of Xanthomonas oryzae pv. oryzicola Suppress AvrXa7-Xa7 Mediated Defense in Rice

15:20

Céline Pesce. Xanthomonas translucens – A Role Of Type III Effectors in Pathogenicity

15:40

Coffee Break

16:00

Walk to the Gold Museum

16:30

Gold Museum visit (guided tour)

18:30

Hotel Transportation

Saturday

July 11th, 2015

6:00

Breakfast

8:00

Hotel-University Transportation

Session VI: Other Pathogenicity Factors Chairwoman: Marie Agnès Jacques 8:50

Yong-Qiang He (Invited speaker) Construction and Analysis of a Transcriptional Regulatory Network of Main Pathogenicity-Regulatory Genes in Xanthomonas campetris pathovar campetris

9:30

Nicolas Chen. Searching for Determinants of Host Specificity in X. axonopodis pv. phaseoli and X. fuscans subsp. fuscans

9:50

Laís Granato. ATP-Dependent RNA Helicase HRPB is Involved in mRNA Regulation of Type IV Operon in Xanthomonas citri subsp. Citri

10:10

Stéphane Cociancich. Full Elucidation of the Hitherto Unknown Structure of Albicidin, a Potent Antibiotic Produced by Xanthomonas albilineans

10:30

Coffee Break

Session VII: Plant Defense Activation and Suppression Chairwoman: Valérie Verdier 11:00

Mary Beth Mudgett (Invited speaker) Tomato 14-3-3 Proteins: Signaling Components of Immunity and Common Targets of Xanthomonas Effectors

11:40

Laurent D. Noël. An Adaptor Kinase Confers Expanded Recognition Specificity to a Plant NLR

12:00

Lunch

Session VIII: Novel Disease Resistance and Control Chairman: Paul Chavarriaga 13:30

Bing Yang (Invited speaker) TALEN- and CRISPR-Mediated Genetic Variations Lead to Resistance to Bacterial Blight in Rice

14:10

Ana Bossa-Castro. Novel Resistance Sources for Bacterial Blight and Bacterial Leaf Streak of Rice in West Africa

14:30

Paula Díaz-Tatis. Engineering Resistant Cassava (Manihot esculenta, Crantz) Plants Against Cassava Bacterial Blight

14:50

Mathilde Hutin. Informed Discovery of xa40(T), a Novel OsSWEET14 Allele from Wild Rice Conferring Broad-Spectrum Resistance to Bacterial Blight of Rice

15:10

Boris Szurek. A Rice Blight Resistance Gene Confers Resistance Against BLB and BLS

15:30

Coffee Break

15:50

Roundtable discussion

16:40

University - Hotel Transportation

17:50

Hotel-Andrés D.C. Transportation

18:50

Closing event in Andres D.C.* *2 possible return times from Andrés D.C (10 p.m. & 1 a.m.)

TABLE OF CONTENTS

Relevant information

17

Abstracts for oral presentations

19

Opening lecture O1-O

Dean Gabriel. Blights, Cankers, Spots, Toxins and Dissemination of Xanthomonas

Session I

Diversity and Diagnosis

20



O2-I

Olivier Pruvost. Tandem Repeats as Understanding the Genetic Structure Xanthomonas citri pv. citri

Genotyping Tools for and Epidemiology of

21

O3-I

Taca Vancheva. Genotyping of Xanthomonas euvesicatoria Strains Using Multiple-Locus Variable Number Tandem Repeat Analysis

22

O4-I

Bart Cottyn. The Diversity of Xanthomonas axonopodis pv. dieffenbachiae Demands a Taxonomic Revision for Accurate Diagnosis

O5-I

Michael Gétaz. Improving Detection of Strawberry Plant Material Infected by the Quarantine Bacterium Xanthomonas fragariae: Development of a LAMP Assay as Diagnostic Tool for On-Field Specific Identification

O6-I

Valérie Verdier. The Power of Genomics in Xanthomonas Diagnostics and Taxonomy

Session II O7-II O8-II

O9-II

23

24

25

Epidemiology and Ecology

Jan Leach. Coping With Combined Stresses: Rice, Global Warming, and Bacterial Disease

26

Déborah Merda. Xanthomonas arboricola Fits the Epidemic Bacterial Population Model with Epidemic Clones Superimposed Upon a Recombinant Network

27

Marion Fischer-Le Saux. Evolution and Comparative Genomics of Pathogenic and Nonpathogenic Strains of Xanthomonas arboricola Unveil Evolutionary Mechanisms of Pathoadaptation

28



Session III

Pathogenesis and Regulation

O10-III

Nian Wang. Exploration of the Post-Transcriptional Regulation of T3SS Genes in Xanthomonas citri

29

O11-III

Ralf Koebnik. Yes We Can – Cannabis as a Promising Host to Study Pathogenicity of Xanthomonads

30

O12-III

Lena Hersemann. Pathogenomics of Xanthomonas translucens pv. graminis to Unravel its Virulence on Forage Grasses

31

O13-III

Cornelius Schmidtke. RNA-Mediated Regulation of Virulence of Xanthomonas campestris pv. vesicatoria

32

O14-III

Mathieu Arlat. CUT Systems of Xanthomonas and PUL Systems of Bacteroidetes Gut Symbionts: A Convergent Evolution to Exploit Plant Compounds?

33

O15-III

Marie Agnès Jacques. A Single Chemotaxis Sensor Defines Xanthomonas campestris Host Range and Restrict it to Brassicaceae.

34

O16-III

Chenyang He. A Novel Cyclic di-GMP Signalling Pathway Mediated by the PdeR-Trip Interaction in Regulation of Virulence of Xanthomonas oryzae pv. oryzae

35



Session IV

Xop Effector Biology

O17-IV

Ulla Bonas. Type III Effectors of Xanthomonas campestris pv. vesicatoria

36

O18-IV

Suayib Üstün. Interplay Between SA Signaling and Xanthomonas Type III Effector Xopj During its Virulence and Avirulence Function

37

O19-IV

César Medina. Characterization of Type Three Effectors from Xanthomonas axonopodis pv. manihotis in Virulence and Suppression of Plant Innate Immunity

38

O20-IV

Rebecca Bart. The Role of the Environment in Host Specificity, Pathogen Virulence and Disease Spread

39

O21-IV

Guido Sessa. Identification and Functional Analysis of Novel Xanthomonas euvesicatoria Type III Effectors

40



Session V

TAL Effector Biology

O22-V

Adam J. Bogdanove. Moderately High-Throughput Xanthomonas Genome Sequencing for Population-Level TAL Effector Analysis

41

O23-V

Alvaro Pérez-Quintero. QueTAL: A Suite of Tools to Classify and Compare TAL Effectors Functionally and Phylogenetically

42

O24-V

Lionel Gagnevin. Which Effectors in the TAL Arsenal of Xanthomonas citri pv. citri are Responsible for Host Specificity on Citrus?

43

O25-V

Gong-You Chen. Transcriptional Activator-Like Effectors TAL6 and TAL11a of Xanthomonas oryzae pv. oryzicola Suppress AvrXa7Xa7 Mediated Defense in Rice

O26-V

Céline Pesce. Xanthomonas translucens – A Role Of Type III Effectors in Pathogenicity

Session VI

44 45

Other Pathogenicity Factors

O27-VI

Yong-Qiang He. Construction and Analysis of a Transcriptional Regulatory Network of Main Pathogenicity-Regulatory Genes in Xanthomonas campetris pathovar campetris

46

O28-VI

Nicolas Chen. Searching for Determinants of Host Specificity in X. axonopodis pv. phaseoli and X. fuscans subsp. fuscans

47

O29-VI

Laís Granato. ATP-Dependent RNA Helicase HRPB is Involved in mRNA Regulation of Type IV Operon in Xanthomonas citri subsp. Citri

48

Stéphane Cociancich. Full Elucidation of the Hitherto Unknown Structure of Albicidin, a Potent Antibiotic Produced by Xanthomonas albilineans

49

O30-VI

Session VII

Plant Defense Activation and Suppression

O31-VII

Mary Beth Mudgett. Tomato 14-3-3 Proteins: Signaling Components of Immunity and Common Targets of Xanthomonas Effectors

50

O32-VII

Laurent D. Noël. An Adaptor Kinase Confers Expanded Recognition Specificity to a Plant NLR

51

Session VIII

Novel Disease Resistance and Control

O33-VIII

Bing Yang. TALEN- and CRISPR-Mediated Genetic Variations Lead to Resistance to Bacterial Blight in Rice

52

O34-VIII

Ana Bossa-Castro. Novel Resistance Sources for Bacterial Blight and Bacterial Leaf Streak of Rice in West Africa

53

O35-VIII

Paula Díaz-Tatis. Engineering Resistant Cassava (Manihot esculenta, Crantz) Plants Against Cassava Bacterial Blight

54

O36-VIII

Mathilde Hutin. Informed Discovery of xa40(T), a Novel OsSWEET14 Allele from Wild Rice Conferring Broad-Spectrum Resistance to Bacterial Blight of Rice

O37-VIII

Boris Szurek. A Rice Blight Resistance Gene Confers Resistance Against BLB and BLS

ABSTRACTS

55 56

For poster presentations

P1-I

Carolina Flores. Characterization of Cassava Bacterial Blight in Venezuela: Diversity of Xanthomonas axonopodis pv. manihotis (Xam) and its Talome

57

P2-I

Paula Martins. Toxin-Antitoxin Distribution and Adaptive Aspects on Xanthomonas Genomes: Focus on Xanthomonas citri

58

P3-I

Isabelle Pieretti. Whole-Genome Sequencing of 18 Strains Reveals High Polymorphism in the Lipopolysaccharides Locus of Xanthomonas albilineans

59

Leidy Rache. Analysis of the Population Diversity of Xanthomonas axonopodis pv. manihotis (Xam) in Different Regions of Colombia Using VNTRS

60

P4-I

P5-I

María Ines Siri. Multilocus Sequence Analysis Reveals High Diversity Among Xanthomonas Strains Affecting Tomato Crops in Uruguay.

P6-II

Michael Gétaz. Phylogeography and Population Structure of Xanthomonas fragariae to Identify Sources and Pathways of Bacterium Through Plant Material Trade

61

62

P7-III

María Victoria Barcarolo. Study of XacPNP in Xanthomonas citri subsp. citri

63

P8-III

Vivian Bernal-Galeano. First Approach to the Discovery of Small Non-Coding RNAs in Xanthomonas axonopodis pv. manihotis

64

P9-III

David Botero. Molecular Pathogenicity Networks in Xanthomonas axonopodis pv. manihotis

65

P10-III

Aude Cerutti. Importance of Hydathodes in Controlling Brassicaceae Vascular Immunity to Xanthomonas

66

P11-III

Fabio Gomez-Cano. Reconstruction of Genome-Scale Metabolic Networks of Species of The Genus Xanthomonas with Different Pathogenic Behavior

P12-IV

Laís Granato. Mining Candidate Effectors in the Xanthomonas citri subsp. citri Genome

P13-V

Nicholas J. Booher. Complete, Single Molecule, Real-Time (SMRT) Sequencing of Xanthomonas oryzae Strains Reveals Complex TAL Effector Gene Relationships

67 68

69

P14-V

Kevin L. Cox Jr. Mechanisms of TAL Effector-Mediated Resistance and Susceptibility to Bacterial Blight of Cotton

70

P15-V

Nargues Falahi Charkhabi. Complete Genome Sequence of the Xanthomonas translucens pv. undulosa Strain ICMP11055

71

P16-V

Zhi-Yuan Ji. A TAL-Free Mutant of Xanthomonas oryzae pv. oryzae Reveals the Essence of TAL Effectors in Bacterial Blight of Rice

72

P17-V

Andrew C. Read. Can Paralogs Subsititute for the Rice Bacterial Leaf Streak Susceptibility Gene OsSULTR3;6?

73

P18-V

Tu Tran Tuan. Functional Analysis of the Talome of African Xanthomonas oryzae pv. oryzae Reveals a New Bacterial Leaf Blight Susceptibility Gene Candidate

74

P19-V

Li Wang. Rice OsSULTR3;6 Functions Quantitatively as a Rice Bacterial Leaf Streak Susceptibility Gene

75

P20-V

Carlos Zárate. How Diverse are TAL Effectors from Xanthomonas axonopodis pv. manihotis Strains in Colombia?

76

P21-VI

Nathaly Montenegro. The Type VI Secretion System of Xanthomonas axonopodis pv. manihotis is Involved in Virulence and In Vitro Motility

P22-VII

P23-VIII

P24-VIII

Dee Dee Luu. Type I Secretion of a Protein Required for Activation of Rice XA21-Mediated Immunity to Xanthomonas oryzae Solange Benítez. In Vivo Evaluation of a Product of Fermented Millet Bacillus subtilis ATCC 55033 for as Potential In Gulupa (Passiflora edulis, Sims) Bacterioses Biocontrol Simone Picchi. N-Acetylcysteine (NAC) Disrupts Biofilm Formation on Xanthomonas citri subsp. citri thus Enhancing Copper Effect

77 78

79

80

Directory of participants

81

Touristic information

85

Brief recommendations

92

Relevant

Information

Conference place: Auditorium LL, Lleras Building. Universidad de los Andes. Bogotá, Colombia. Address: Cr. 1 No. 18A-12. Postal code: 111711. GPS (google maps): 4.602481, - 74.065090 Phone: (+57 1) 3394949 Hotel IBIS Bogotá Museo Transversal 6 No 27 – 85. Centro Internacional. Bogotá, Colombia. Phone: (+57 1) 3814666 General information: [email protected] GPS: N 4° 36’ 51.03’’ W 74° 4’ 7.66’ Contact phones: Contact us for any question or indication to: (+57 1) 3394949 ext. 3492 * from a local cell phone, dial 031 instead of +57 1 Adriana Bernal: +57 320 2971464* Vivian Bernal: +57 3014654052* * from a local cell phone, dial without the +57 Contact email: [email protected] Emergency number: 123 Internet connection Wifi network : EventosUAndes Key: UAeventos2015

18

ABSTRACTS FOR ORAL PRESENTATIONS

Opening lecture

O1-O Opening lecture

Blights, Cankers, Spots, Toxins and Dissemination of Xanthomonas

Dean W. Gabriel

Plant Pathology Dept. & Plant Molec. and Cell Biology Program, University of Florida, Gainesville, FL USA. Xanthomonas is a surprisingly flexible genus of hemibiotrophs with a very wide host range consisting of species and strains that exhibit a high level of host specificity. There are well over 140 different pathogenic variants (pathovars) that are typically highly clonal in population structure and differ primarily in host range. Some very different clonal groups (citrus canker, common bean blight) have nearly identical host range and can cause identical diseases, often using plasmid borne or horizontally transferred pathogenicity (pth) genes with extraordinary mutagenic potential. These pth genes encode Type III injected effectors that elicit a wide range of symptoms ranging from hyperplastic cankers to leaf blights, spots, and wilts. Often the symptoms elicited are an escape mechanism used along with xanthan gum to enhance transmission. By contrast with all other pathogenic xanthomonads, X. albilineans, the causal agent of sugarcane leaf scald, does not possesses genes encoding xanthan gum or the hypersensitive response and pathogenicity (Hrp) T3SS, but instead this reduced genome (3.8 Mb) species encodes numerous cell wall degrading enzymes, multiple NRPS (Non Ribosomal Peptide Synthetase) and PKS (Polyketide Synthase) toxin clusters and a Salmonella pathogenicity island-1 (SPI-1) T3SS that is typically considered important only for animal pathogenicity. Mutations of this SPI-1 system appeared unaffected in pathogenicity of sugarcane. The role of quorum sensing and biofilm formation for survival of Xanthomonas will be discussed.

21

Session I: Diversity and Diagnosis

O2-I Keynote talk

Tandem Repeats as Genotyping Tools for Understanding the Genetic Structure and Epidemiology of Xanthomonas citri pv. citri

Alice Leduc1, Yaya. N. Traoré2, Karine Boyer1, Maxime Magne1, Pierre Grygiel1, Jonathan Gordon1, Claudine Boyer1, Fabien Guerin1, Issa Wonni3, Léonard Ouedraogo3, Ralf Koebnik4, Valérie Verdier4, Lionel Gagnevin1,4, Christian Vernière1,5, Virginie Ravigné1,5 and Olivier Pruvost1

UMR PVBMT (CIRAD/Université de la Réunion), F-97410 Saint-Pierre, La Réunion, France 2 Institut Polytechnique Rural, Katibougou, Mali 3 Institut de l’Environnement et de Recherches Agricoles, Bobo Dioulasso 01, Burkina Faso 4 UMR IPME (IRD/CIRAD/Université de Montpellier), F-34394 Montpellier, France 5 UMR BGPI (INRA/CIRAD/Montpellier SupAgro), F-34398 Montpellier, France 1

MultiLocus Variable number of tandem repeat Analysis (MLVA) has been extensively used to examine epidemiological and evolutionary issues on monomorphic human pathogenic bacteria. MLVA is gaining popularity on agriculturally important bacterial plant pathogens as a tool to improve our understanding of their epidemiology. Xanthomonas citri pv. citri, the causal agent of Asiatic citrus canker, is a quarantine organism in several countries and a major threat for the citrus industry worldwide. We screened the genomes of X. citri pv. citri strain IAPAR 306 and of phylogenetically related strains for tandem repeats. Two MLVA schemes targeting minisatellites and microsatellites, respectively, were developed to assess the diversity of this monomorphic bacterium at various spatio-temporal scales. Microsatellites are useful for outbreak investigation. In contrast, minisatellites were used to decipher the global diversity of X. citri pv. citri and revealed four pathotype-specific lineages based on Discriminant Analysis of Principal Components (DAPC). A single lineage (DAPC1) comprised strains that were implicated in the major geographical expansion of X. citri pv. citri during the 20th century. When applied to the analysis of the emergence of in Africa, minisatellites revealed the presence of two different genetic lineages in addition to DAPC1 on this continent, consistent with SNP data derived from draft genome sequences. Using bacterial populations collected in two neighboring Western African countries, Mali and Burkina Faso, we applied these two genotyping schemes to investigate the origin and pathways of these emergences. Minisatellite and SNP data suggested the introduction of two groups of strains in Mali (DAPC1 and DAPC2). DAPC2 was restricted to Bamako district, whereas DAPC1 strains were found much more invasive. The latter strains formed a major clonal complex based on microsatellite data. This suggests that human activities played a major role in the spread of DAPC1 strains via the movement of contaminated propagative material, further supported by the low differentiation between populations from geographically distant nurseries and orchards and the fact that many nursery strains were assigned to the primary or secondary founder haplotypes identified in the minimum spanning tree. Approximate Bayesian Computation analyses supported the hypothesis that strains from Burkina Faso resulted from a bridgehead invasion from Mali. The new minisatellite scheme represents an opportunity for international X. citri pv. citri genotyping and data sharing. The data generated in this study was deposited in the Xanthomonas citri genotyping database (http://www.biopred.net/MLVA/). 22

Session I: Diversity and Diagnosis

O3-I

Genotyping of Xanthomonas euvesicatoria Strains Using MultipleLocus Variable Number Tandem Repeat Analysis

Taca Vancheva1,2, Mariya Stoyanova3, Nevena Bogatsevska3, Penka Moncheva1, Ralf Koebnik2

Faculty of Biology, Sofia University St. Kliment Ohridski, Sofia, Bulgaria UMR Interactions Plantes Microorganismes Environnement, IRD-Cirad-UM, Montpellier, France 3 Institute of Soil Science, Agrotechnologies and Plant Protection Nikola Poushkarov, Sofia, Bulgaria 1 2

Bacterial spot (BS) of pepper caused by the pathogens of genus Xanthomonas is an economically important disease affecting pepper plantations all over the world. During the last 20 years, the causative agents of BS have been subject to many reclassifications. According to the current data, BS is caused by four widely distributed species: Xanthomonas euvesicatoria (Xeu), Xanthomonas vesicatoria (Xv), Xanthomonas perforans, and Xanthomonas gardneri. Two Xanthomonas species (Xeu, Xv) are identified as causal agents of BS in Bulgaria and Macedonia. A profound knowledge of the pathogen population structure is necessary for efficient control of BS in different geographical regions. Diversity of BS agents has been studied by phenotypic, biochemical and DNA-based methods. However, a precise molecular typing tool for diagnostic and differentiation is essential for monitoring the population structure and dynamics. To assess the genetic diversity of 96 Xeu strains from the Balkan Peninsula we sequenced the genomes of two Xeu strains and developed a multilocus variable-number tandem repeat analysis (MLVA) scheme. In total, 16 tandem repeat markers were used to screen for typeability and polymorphism between the Xeu strains. The number of alleles per locus ranged from 1 to 16. Combining all 16 loci into a MLVA-16 scheme resulted in an Hunter Gaston Discriminatory Index (HGDI) of 0.83, thus demonstrating their suitability for epidemiological surveillance of Xeu strains. Latest results on the structuration of Xeu populations from Bulgaria and Macedonia will be presented. Acknowledgements: This study was supported by the National Science Research Fund, Bulgaria by contract No ДФНИ-Б02/4.

23

Session I: Diversity and Diagnosis

O4-I

The Diversity of Xanthomonas axonopodis pv. dieffenbachiae Demands a Taxonomic Revision for Accurate Diagnosis

E.C. Constantin1, I Cleenwerck2, M. Maes1, S. Baeyen1, C. Van Malderghem1, P. De Vos2, B. Cottyn1

Plant Sciences Unit - Crop Protection, Institute for Agricultural and Fisheries Research (ILVO), Burg. Van Gansberghelaan 96, Merelbeke, Belgium. 2 BCCM/LMG Bacteria Collection – Faculty of Science, Ghent University, K.L. Ledeganckstraat 35, Ghent, Belgium 1

Xanthomonas axonopodis pv. dieffenbachiae (Xad) is the causal agent of anthurium bacterial blight and listed as an A2 quarantine organism by EPPO. In support of plant health policies, QBOL generated barcodes for rapid identification of regulated Xanthomonas, including Xad. Xad strains as well as strains from closely related pathovars were collected, consisting of pathotype and well characterized strains from different hosts, geographic origin and isolation year. Strains named as Xad did not represent a single biological entity and were further examined using a polyphasic approach including multilocus sequence analysis (MLSA), DNA-DNA hybridization (DDH), calculations of average nucleotide identity (ANI) values, FAME, Biolog and pathogenicity tests on five aroid hosts. MLSA of seven genes showed Xad strains to cluster into three phylogenetic groups (PG I, II and III) that largely correspond with host from which isolated. Based on ANIb values, DDH data, and phenotypic characteristics, the three PGs each belong to a different species. Taxonomic proposals involve: emendations of existing species and the description of X. dieffenbachiae sp. nov. as a new species, resulting in the classification of Xad strains from PG I, II and III, as X. citri pv. aglaonemae pv. nov., X. euvesicatoria pv. philodendri pv. nov., and X. dieffenbachiae pv. dieffenbachiae sp. nov. comb. nov., respectively. Whole-genome comparative analysis for virulence related factors did not reveal the pathogenic variation among strains observed after host inoculations. The proposed new classification has consequences for several quarantine organisms on the EPPO list.

24

Session I: Diversity and Diagnosis

O5-I

Improving Detection of Strawberry Plant Material Infected by the Quarantine Bacterium Xanthomonas fragariae: Development of a LAMP Assay as Diagnostic Tool for On-Field Specific Identification

Michael Gétaz1, Joël F. Pothier1, Andreas Bühlmann2, Pierre H.H. Schneeberger2,3, Cinzia Van Malderghem4, Bart Cottyn4, Martin Maes4, Brion Duffy1

Zürich University of Applied Sciences ZHAW, Institute of Natural Resource Sciences, Environmental Genomics and Systems Biology research group, Wädenswil, Switzerland. 2 Agroscope Changins-Wädenswil ACW, Department of Epidemiology and Molecular Diagnostics, Wädenswil, Switzerland. 3 Swiss tropical and Public Health Institute, Institute of Epidemiology and Public Health, Ecosystem Health Sciences, Basel, Switzerland. 4 Institute for Agricultural and Fisheries Research ILVO, Crop Protection, Marelbeke, Belgium 1

Xanthomonas fragariae is a quarantine Gram-negative bacterium causing angular leaf spot of strawberry, which can be particularly severe under protected cultivations with high density plots aided by high humidity and sprinkler irrigation systems. Its first apparition was observed in the USA in 1960 and was already considered as a potential severe disease. In recent decades, globalization has increased international movement of plants and plant material through trade and human travels, which influence spread of pests and pathogens. Detection of X. fragariae could be achieved by visual inspection when distinct symptoms are present but since different Xanthomonas can be found on strawberries with close relativeness, molecular techniques are then required for a reliable identification. Furthermore, bacterial spread can be helped by trade of symptomless infected strawberry and therefore these require a method allowing detection of latent invisible infections to prevent spread of infected material. We developed a reliable, quick and easy loop-mediated isothermal amplification (LAMP) assay for specific detection of X. fragariae. This detection method was retained for its rapidity, efficiency, and specific on-field amplification of DNA sequences under isothermal conditions not relying on expensive and sophisticated thermal cycling instruments. On-field application can therefore help containing bacterial spread by providing early detection of the pathogen even by low-experienced staff. LAMP primer set was designed using a “dualBLAST” pipeline performed on all complete genomes from selected organisms of interests to find the most conserved and specific molecular marker to discriminate X. fragariae from other Xanthomonas species and pathovars.

25

Session I: Diversity and Diagnosis

O6- I

The Power of Genomics in Xanthomonas Diagnostics and Taxonomy

Valérie Verdier

IRD, Cirad, Univ. Montpellier, Interactions Plantes Microorganismes Environnement (IPME), 34394 Montpellier, France Molecular diagnostics for crop diseases enhance food security because they enable rapid identification of threatening pathogens and provide critical information for deployment of disease management strategies. Comparative genomics has facilitated the development of diagnostics tools including multiplex PCR and loop mediated isothermal amplification (LAMP) for field and quarantine laboratory diagnosis of rice bacterial diseases. These tools can be adapted, when genomic sequence is available, to differentiate pathovars or lineages of Xanthomonas sp. High throughput, sensitive and highly specific diagnostics can now streamline field to farmer information on disease occurrence as well as how populations evolve in geographically distinct areas. Genome sequence has also help to identify new species of Xanthomonas and to clarify the taxonomic position of strains of X. oryzae isolated on weeds. Current works using Xanthomonas draft genome sequence to develop diagnosis and to study the genetic relatedness of X. oryzae strains will be discussed.

26

Session II: Epidemiology and Ecology

O7-II Keynote talk

Coping With Combined Stresses: Rice, Global Warming, And Bacterial Disease

Jan E. Leach

Colorado State University, Plant Sciences Bldg, Fort Collins, CO USA 805231177 Warming in the climate system presents a major challenge for scientists focused on agricultural production systems because many plant diseases are predicted to intensify as environmental temperatures increase. The problem of increased disease pressure is compounded by the fact that many widely used single gene sources of disease resistance (R genes) are less effective at high temperatures. While the phenomenon of temperatureinduced susceptibility is well documented, the mechanisms are not known. In rice, high temperatures are conducive to bacterial blight (BB) disease. Furthermore, most R genes are less effective at controlling disease at high relative to low temperature regimes. The exception is the Xa7 BB R gene, which provides increased resistance to Xanthomonas oryzae pv. oryzae at high relative to low temperatures. Transcriptome and phenotypic responses in the rice-BB system are providing intriguing insights into the impacts of high temperature on plant-bacterial interactions. The long term goal of our studies is to understand how plants respond to multiple stresses, information that will help in designing strategies to mitigate the negative impacts of climate changes on cropping systems.

27

Session II: Epidemiology and Ecology

O8-II

Xanthomonas arboricola Fits the Epidemic Bacterial Population Model with Epidemic Clones Superimposed Upon a Recombinant Network

Déborah Merda1,2,3, Sophie Bonneau1,2,3, Jean-François Guimbaud1,2,3, Christophe Lemaire1,2,3, Marie-Agnès Jacques1,2,3, Marion Fischer-Le Saux1,2,3

INRA, UMR 1345 IRHS, Beaucouzé, France AGROCAMPUS OUEST, UMR 1345 IRHS, Beaucouzé, France 3 Université d’Angers, UMR 1345 IRHS, SFR4207 Quasav, PRES L’UNAM, Beaucouzé, France 1 2

Disease emergence is a major problem in agroecosystems. Acquisition of virulence factors through horizontal gene transfers (HGT) can modify the pathogenic profile of strains and lead to the emergence of new diseases. Within bacteria, HGTs are more frequent for individuals which are phylogenetically close. As pathogenic and nonpathogenic individuals can coexist within the same species, it is very likely that they exchange genetic material when in sympatry. Nonpathogenic strains are defined as strains, which are unable to cause disease on their host of isolation. In order to understand pathogen emergence in agroecosystems, it is important to identify the evolutionary mechanisms, which led to the installation of these two types of populations. The species Xanthomonas arboricola encompasses nine pathovars (an infrasubspecific division grouping strains causing the same disease on the same host range) as well as nonpathogenic strains. We performed a population genetic analysis using 188 strains with seven housekeeping genes, and we analyzed the evolution of the virulence-associated genes repertoires in these strains. X. arboricola presents an epidemic structure according to the Maynard Smith’s definition (2000). The three highly aggressive pathovars (X. arboricola pv. juglandis, X. arboricola pv. pruni and X. arboricola pv. corylina) represent epidemic clones whose emergence is linked to the acquisition of nine type three effectors. Nonpathogenic strains and strains from minor pathovars of this species represent the recombinant network within which loss of virulence-associated factors is evidenced for nonpathogenic strains. Genomic and phenotypic analyses will improve our knowledge of the ecological niche of nonpathogenic strains. Reference: Maynard Smith, J., Feil, E.J., Smith, N.H., 2000. Population structure and evolutionary dynamics of pathogenic bacteria. Bioessays 22, 1115– 1122.

28

Session II: Epidemiology and Ecology

O9-II

Evolution and Comparative Genomics of Pathogenic and Nonpathogenic Strains of Xanthomonas arboricola Unveil Evolutionary Mechanisms of Pathoadaptation

Marion Fischer-Le Saux1,2,3, Sophie Cesbron1,2,3, Martial Briand1,2,3, Salwa Essakhi1,2,3, Sophie Gironde1,2,3, Charles Manceau1,2,3, Marie-Agnès Jacques1,2,3 1 2 3

INRA, UMR1345 IRHS, Beaucouzé, F-49070, France AGROCAMPUS-OUEST, UMR1345 IRHS, Beaucouzé, F-49070, France UNIVERSITE D’ANGERS, UMR1345 IRHS, Beaucouzé, F-49070, France

Xanthomonas arboricola pv. juglandis (Xaj) is the causal agent of walnut blight (WB), a disease affecting walnut production worldwide and of vertical oozing canker (VOC), a new disease that emerged in French walnut orchards in the early 2000s. In addition, nonpathogenic X. arboricola strains were extensively isolated from healthy and diseased walnut samples. We used multilocus sequence analysis and multilocus variable-number tandem repeat analysis on a collection representing the diversity of X. arboricola to unveil its genetic structure. We showed that Xaj strains grouped in a clonal complex within which VOC strains represent specific sequence types. Nonpathogenic strains constituted distant clusters clearly distinct from the Xaj clonal complex. In order to decipher the genes and functions responsible for pathoadaptation, as well as evolutionary mechanisms involved, we sequenced the genomes of two pathogenic strains (CFBP 2528 associated to WB and CFBP 7179 associated to VOC) and two nonpathogenic strains (CFBP 7634 and CFBP 7651). Among the most striking features revealed by comparative genomics was the difference in transposase content. Only four transposases were predicted in the genomes of the nonpathogenic strains whereas ten times more were present in pathogenic strains. Genome sequencing also revealed the presence of a 95 Kb genomic island encoding genes responsible for copper, acriflavin and heavy metal resistance in the VOC strain. Its presence correlated with the copper resistance of this strain and we showed that this genomic island is widespread in recent VOC isolates. Mobile elements play a crucial role in ecological adaptation of X. arboricola

29

Session III: Pathogenesis and Regulation

O10-III Keynote talk

Exploration of the Post-Transcriptional Regulation of T3SS Genes in Xanthomonas citri

Maxuel O. Andrade1, Chuck S. Farah2, Nian Wang1*

Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, FL, 33850 USA. 2 Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil. * Correspondence E-mail: [email protected] 1

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a devastating disease of most commercial citrus varieties causing huge economic losses worldwide. Type III secretion system (T3SS) and type III effectors are required for the pathogenicity of Xcc. Here, we present our recent study on regulation of the T3SS genes by RsmA/CsrA in Xcc. The RsmA/CsrA family of post-transcriptional regulators of bacteria are involved in the regulation of many cellular processes, including pathogenesis. We have found that rsmA is required for the hypersensitive response (HR) and pathogenicity of Xcc. Microarray, quantitative reverse transcription PCR, western-blotting, and GUS assay analyses indicated that RsmA regulates the expression of T3SS genes at both transcriptional and post-transcriptional levels. We found that RsmA activates expression of hrp/hrc genes encoding T3SS by directly binding to the 5’ untranslated region (UTR) of hrpG, the master regulator of hrp/hrc genes in Xcc. Our experiments revealed that RsmA stabilizes hrpG mRNA, leading to increased HrpG protein levels and hrp/hrc gene activation. The activation of hrp/hrc genes by RsmA via HrpG was further supported by the observation that ectopic overexpression of hrpG in an rsmA mutant restored its pathogenicity in host plants and HR in non-host plants. Our results further indicate that RsmA controls hrpG mRNA stability but not the signaling pathway involving its phosphorylation. Taken together, these data reveal that RsmA primarily activates T3SS by acting as a post-transcriptional regulator of hrpG and that this regulation is critical to mediate pathogenicity of Xcc.

30

Session III: Pathogenesis and Regulation

O11-III

Yes We Can – Cannabis as a Promising Host to Study Pathogenicity of Xanthomonads

Jonathan M. Jacobs1, Céline Pesce1,2, Pierre Lefeuvre3, Ralf Koebnik1

UMR Interactions Plantes Microorganismes Environnement, IRD-Cirad-UM, Montpellier, France 2 Earth and Life Institute, Applied Microbiology Phytopathology, Université Catholique de Louvain la Neuve, Belgium 3 Cirad, UMR PVBMT, Pôle de Protection des Plantes, Saint-Pierre, Ile de la Réunion, France 1

Pathogenic bacteria in the genus Xanthomonas collectively cause disease on many host plants, including Cannabis sativa L. (cannabis). We sequenced the genomes of two strains that were previously isolated from symptomatic hemp plants in Romania and Japan. Comparative multilocus sequence analyses and genome-wide sequence comparisons suggest that the two cannabis strains belong to the same species as the recently isolated bean-pathogenic strain Nyagatare. Interestingly, the cannabis strains do not possess a type III secretion system (T3SS), a major virulence secretion system, nor type IIIsecreted virulence effectors found in many Xanthomonas spp. Most notably, the genomes contained genes encoding the T3SS regulators HrpG and HrpX. Promoter prediction of HrpX-regulated genes suggests that HrpX regulates the expression of type II-secreted hydrolytic enzymes, which are similarly found in other plant-pathogenic Xanthomonas strains. The genome of a distantly related rice isolate, Xanthomonas maliensis strain 97M, similarly lacks the T3SS but encodes HrpG, HrpX and putatively HrpX-regulated hydrolytic enzymes. This is consistent with the current hypothesis that HrpG and HrpX regulate pathogenesis beyond the T3SS alone. Intriguingly, the closely related Nyagatare strain possesses a T3SS and associated effectors. We propose a stepwise evolution of pathogenicity in Xanthomonas spp. where strains acquire: 1) pathogenicity regulators HrpG and HrpX, 2) the T3SS, and 3) type III-secreted effector genes. We will provide hypotheses about the molecular interplay between the cannabis pathogen and its host and non-host plants.

31

Session III: Pathogenesis and Regulation

O12-III

Pathogenomics of Xanthomonas translucens pv. graminis to Unravel its Virulence on Forage Grasses

Lena Hersemann1, Franco Widmer1, Frank-Jörg Vorhölter2, Roland Kölliker1

Institute for Sustainability Sciences ISS, Agroscope, Reckenholzstrasse 191, 8046 Zurich, Switzerland 2 CeBiTec, Bielefeld University, Universitaetsstrasse 27, 33615 Bielefeld, Germany 1

Bacterial wilt is one of the most serious diseases of temperate forage grasses, such as Italian ryegrass, leading to significant losses in yield and quality. The disease is caused by X. translucens pv. graminis (Xtg), a gram-negative bacterium that enters the plants primarily through wounded tissue, multiplies in intercellular spaces and spreads through the xylem. Infected, susceptible plants wilt within a few days after infection. A detailed understanding of the genetic control of this complex host-pathogen interaction is indispensable for the further development of Italian ryegrass cultivars with increased resistance to bacterial wilt and to refine and optimize breeding procedures. In order to gain further insight into the underlying pathogenesis mechanisms, factors influencing pathogen virulence were investigated based on whole genome sequencing. Comparative analysis with other Xanthomonads revealed a noncanonical T3SS as a striking characteristic of this pathovar and analysis of additional Xtg strains from Switzerland, Norway and New Zealand confirmed swarming-deficiency as well as the absence of TAL effectors as common traits. Even if Xtg appears to possess a reduced set of virulence factors, comparative genome analysis with Xanthomonas spp., shown to be non-pathogenic on Italianryegrass, revealed two effector proteins to be exclusively presented in the pathovar graminis. In a next step targeted knock-out mutagenesis of both effectors will be performed to verify their function as crucial virulence factors for causing bacterial wilt in Italian ryegrass.

32

Session III: Pathogenesis and Regulation

O13-III

RNA-Mediated Regulation of Virulence of Xanthomonas campestris pv. vesicatoria

Cornelius Schmidtke1, Juliane Brock1, Ulrike Abendroth1, Javier Serrania2, Doreen Blüher1, Ivo Große3, Anke Becker2 and Ulla Bonas1

Institute for Biology, Dept. of Genetics, Martin-Luther-Universität HalleWittenberg, D-06099 Halle, Germany 2 Loewe Center for Synthetic Microbiology and Department of Biology, Philipps-Universität Marburg, Germany 3 Institute for Informatics, Dept. of Bioinformatics, Martin-Luther-Universität Halle-Wittenberg, D-06099 Halle, Germany 1

Pathogenicity of Xanthomonas campestris pv. vesicatoria (Xcv) relies on the type III secretion system (T3SS), which delivers effector proteins directly into the plant cell. We recently discovered more than 20 small noncoding RNAs (sRNAs) in Xcv strain 85-10, some of which are co-regulated with the T3SS (1). Deletion and complementation studies revealed that the constitutively expressed sRNA sX13 promotes Xcv virulence and represents a novel RNA-regulator of the T3SS (2). The sX13 regulon comprises more than 60 genes and suggests a central role of sX13 in the regulation of signal transduction, motility and virulence. sX13 represses translation of target mRNAs depending on C-rich sX13 loops and complementary G-rich mRNA-motifs, which appear to serve as translational enhancers. To address the role of RNA-binding proteins in Xcv virulence, we focused on Hfq (host factor for phage Qβ RNA replication) and CsrA1 (carbon storage regulator A). The RNA-chaperone Hfq mediates sRNA-mRNA interactions in many bacteria. Members of the CsrA/RsmA protein family mostly act as translational repressors. While inactivation of hfq did not affect Xcv virulence (2), csrA1 mutants are non-pathogenic and exhibit pleiotropic phenotypes. Since the activity of CsrA/RsmA family members is regulated by binding of specific classes of sRNAs, which are unknown in Xanthomonas spp., we aimed at the experimental identification of these sRNAs. Using co-immunoprecipitation, RNA-Seq and in vitro interaction assays we confirmed direct binding of three sRNAs to CsrA1. References: 1. Schmidtke, C., Findeiß, S., Sharma, C.M., Kuhfuss, J., Hoffmann, S., Vogel, J., Stadler, P.F. and Bonas, U. (2012) Genome-wide transcriptome analysis of the plant pathogen Xanthomonas identifies sRNAs with putative virulence functions. Nucleic Acids Res 40: 2020-2031. 2. Schmidtke, C., Abendroth, U., Brock, J., Serrania, J., Becker, A. and Bonas, U. (2013) Small RNA sX13: a multifaceted regulator of virulence in the plant pathogen Xanthomonas. PLoS Pathog 9: e1003626. 33

Session III: Pathogenesis and Regulation

O14-III Invited talk

CUT Systems of Xanthomonas and PUL Systems of Bacteroidetes Gut Symbionts: A Convergent Evolution to Exploit Plant Compounds?

Matthieu Arlat1,2,3, Sébastien Carrère1,2 and Emmanuelle Lauber1,2

INRA, Laboratoire des Interactions Plantes Micro-organismes (LIPM), UMR 441, Castanet-Tolosan, France 2 CNRS, Laboratoire des Interactions Plantes Micro-organismes (LIPM), UMR 2594, Castanet-Tolosan, France 3 Université Paul Sabatier, Toulouse, France 1

Xanthomonas species display an overrepresentation of TBDT genes which code for outer membrane transporters involved in the uptake of iron or carbohydrates. TBDTs involved in carbohydrate transport belong to socalled CUT systems which comprise degradative enzymes, transporters and regulators. This TBDT overrepresentation and organization in CUT systems is a characteristic of bacteria of the Xanthomonadaceae family but also of oligotrophic bacteria belonging to the Caulobacterales. Interestingly, Bacteroidetes isolated from animal or human gut also display TBDT overrepresentation These TBDT genes belong to PUL systems which encode degradative enzymes, transporters and regulators similar to CUT systems. The major difference between PUL and CUT systems lies in TBDTs which belong to very different groups. TBDTs from Bacteroidetes belong to SusC family and are associated with accessory proteins of the SusD family that are not present in CUT systems. A large scale comparative study of CUT and PUL systems suggests a convergent evolution to associate TBDT and plant cell wall-degradation. Moreover, this analysis defines Xanthomonas specific CUT systems, thus shedding light on the evolution of this genus.

34

Session III: Pathogenesis and Regulation

O15-III

A Single Chemotaxis Sensor Defines Xanthomonas campestris Host Range and Restrict it to Brassicaceae

Marie-Agnès Jacques1, Arnaud Indiana1, Martial Briand1, Laurent D. Noël2, and Armelle Darrasse1

INRA, UMR1345 Institut de Recherche en Horticulture et Semences, F-49071 Beaucouzé, France. 2 INRA-CNRS, LIPM UMR441, F-31326 Castanet-Tolosan, France. 1

Deciphering mechanisms shaping bacterial adaptation to host range should help to predict the emergence of- and control- infectious diseases. During the early stages of infection, pathogenic bacteria must enter into host plant tissues to multiply. This process may be mediated by plant signals originating from entry sites. Chemotaxis allows motile bacteria to detect and move toward attractants and away from repellents through detection by cell membrane-bound chemoreceptors, called Methyl-accepting Chemotaxis Proteins (MCPs). To test the contribution of chemotaxis to leaf ingress, the genes encoding a key element of the chemotactic signal transduction system, CheY, and one MCP, XCC0324 renamed Hsb1 for host specificity in brassicas, were mutagenized in Xanthomonas campestris pv. campestris (Xcc) strain ATCC33913, a vascular pathogen of brassicas. While this MCP is found in several lineages of Xanthomonas, a group of alleles of this gene is confined to X. campestris. Groups of divergent alleles are found in phylogenetically closely related strains belonging to several species, none of which being pathogenic on Brassicaceae. Both cheY and hsb1 are required for an efficient ingress into leaf tissues of host plants, such as radish and Arabidopsis. Hsb1 is required for the attraction of Xcc by a wound on cabbage leaves, but is not involved in aggressiveness per se, as observed following injection of Xcc∆hsb1 directly into leaf vasculature. Based on capillary assays Hsb1 confers to Xcc the ability to detect sinigrin and L-phenylalanine. In conclusion, Hsb1 plays a role in the detection of attractive signal(s) leading to Xcc ingress into host leaf tissues by a yet unknown mechanism.

35

Session III: Pathogenesis and Regulation

O16-III

A Novel Cyclic di-GMP Signalling Pathway Mediated by the PdeR-Trip Interaction in Regulation of Virulence of Xanthomonas oryzae pv. oryzae

Haiyun Li,1 Fang Tian,1 Dingrong Xue1, Huamin Chen1, Xiaochen Yuan2, ChingHong Yang2, Chenyang He1

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China 2 Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA 1

PdeK/PdeR, the two-component regulatory system (TCS), was previously shown to regulate the virulence of Xanthomonas oryzae pv. oryzae (Xoo), the bacterial blight pathogen of rice. The response regulator PdeR harbors phosphodiesterase (PDE) activity to degrade bacterial second messenger cyclic di-GMP. To understand the downstream signaling pathway mediated by PdeR, we started with looking for its interacting partners in Xoo strain PXO99A. Here, we identified a response regulator PXO_04421, named as TriP, as an interactor of PdeR. Yeast two-hybrid (Y2H) and GST pull-down assays confirmed the specific PdeR-Trip interaction. Interestingly, the interaction was inhibited by high concentration of c-di-GMP. Virulence assays demonstrated that the triP mutant caused shorter lesion length on rice leaves than the wild type, and its ability to progress through the xylem tissue was also impaired. Moreover, both the pdeR and the triP single mutant produced less exopolysaccharide (EPS), while the pdeR/triP double mutant produced similar level of EPS with that of the triP mutant. These results indicated that TriP and PdeR are functionally related in regulation of bacterial virulence on rice. Additionally, RNA-seq analysis revealed a considerable overlap in the upregulated genes in the two single mutants, which further implied that TriP might mediate a part of the PdeR-mediated signaling in Xoo.

36

Session IV: Xop Effector Biology

O17-IV Keynote talk

Type III Effectors of Xanthomonas campestris pv. vesicatoria

Ulla Bonas

Institute for Biology, Dept. of Genetics, Martin-Luther-Universität Halle-Wittenberg, D-06099 Halle, Germany Pathogenicity of most xanthomonads depends on a type III secretion (T3S) system, a nanomachine that is induced in the plant tissue and which translocates effector proteins (T3Es) into the plant cell cytosol. We study the interaction between Xanthomonas campestris pv. vesicatoria (Xcv) and its host plants pepper and tomato. In susceptible plants, T3Es interfere with host cell processes to the benefit of the pathogen and allow bacterial proliferation in the apoplastic space of the plant leaf mesophyll. In resistant plants, single resistance genes mediate recognition of individual T3Es. T3E recognition often induces a hypersensitive response (HR) of plant cells, a rapid and localized programmed cell death. Xcv injects more than 25 different T3Es into the plant cell, termed Avr (avirulence protein) or Xop (Xanthomonas outer protein). Among the T3Es from Xcv are plant immunity suppressors, cell death inducers, a ubiquitin ligase, a transcription factor and proteins of unknown function. Selected T3Es will be discussed.

37

Session IV: Xop Effector Biology

O18-IV

Interplay Between SA Signaling and Xanthomonas Type III Effector XopJ During its Virulence and Avirulence Function

Suayib Üstün1 and Frederik Börnke1,2

Leibniz-Institute of Vegetable and Ornamental Crops (IGZ), Großbeeren, Germany 2 Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany 1

Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease in pepper and tomato. To overcome the basal defence of plants Xcv translocates about 30 type III effector proteins (T3E) via its type III secretion system into the host cell. These T3Es are able to manipulate host cellular processes involving vesicle trafficking, the ubiquitin/proteasome system (UPS) and gene expression, although most of their plant targets are still unknown. Evidence is emerging that manipulation of the UPS might be an effective and widespread virulence strategy of bacterial invaders to promote pathogenesis. In line with this, we could show that Xcv T3E XopJ, a member of the YopJ family of T3Es, promotes bacterial virulence on susceptible pepper plants through the inhibition of the proteasome and a resultant suppression of salicylic acid (SA) – dependent defense responses. XopJ recruits proteasomal subunit RPT6 to the plasma membrane and acts as a cysteine protease to specifically degrade RPT6. A lack of RPT6 might prevent the assembly of a functional proteasome and thus leads to its malfunction. Consequently, XopJmediated suppression of the proteasome function impairs the proteasomal turnover of NPR1 and hence leads to its accumulation. Intriguingly, besides its virulence function, XopJ triggers a hypersensitive response (HR) and acts as an avirulence factor when plants were exogenously treated with SA. Our results suggest that XopJ-triggered HR-like symptoms are closely related to its virulence function and that XopJ follows a two-signal model in order to elicit a response in the non-host plant N. benthamiana. References [1] Üstün S and Börnke F (2015) Plant Physiol: DOI:10.1104/pp.15.00132 [2] Üstün S and Börnke F (2014) Front. Plant Sci. 5:736. [3] Üstün S, Bartetzko V, Börnke F (2013) PLoS Pathog 9(6): e1003427.

38

Session IV: Xop Effector Biology

O19-IV

Characterization of Type Three Effectors from Xanthomonas axonopodis pv. manihotis in Virulence and Suppression of Plant Innate Immunity

Cesar A. Medina, Juan Luis Gonzalez, Paola Reyes, David Bejarano, Cesar A. Trujillo, and Adriana Bernal.

Laboratorio de Micología y Fitopatología. Universidad de los Andes. Bogotá, Colombia. Xanthomonas axonopodis pv. manihotis (Xam) causes Cassava Bacterial Blight, threatening food security of some of the most vulnerable communities in the tropics. Xam, like other Xanthomonas requires T3Es for pathogenicity. The best sequenced strain, CIO151, has 19 non Transcriptional Activator Like Effector (TALE) T3Es and 2 TALEs. This work is focused on the molecular characterization of 10 non TALE T3Es present in Xam CIO151. Our findings remark the importance of AvrBs2, XopAO1, XopX and XopZ for maximal virulence and a redundant function for XopN and XopQ in the susceptible cultivar MCOL2215. Additionally, we tested PAMP Trigger Immunity (PTI) and Effector Trigger Immunity (ETI) suppression using Arabidopsis as a heterologous system. AvrBs2, XopAO1, XopQ, XopN and XopAE are capable of suppressing callose deposition. ETI suppression activity was only detected for XopAO1 and XopE4. Finally, transient expression using Agrobacterium tumefaciens of different T3Es resulted in HR elicitation by the effector XopAO1 in Nicotiana tabacum and HR elicitation on specific cassava cultivars by XopR and XopE1. This is the first time that HR is reported in different cassava varieties by specific T3Es from Xam. These results showed the importance and diversity in function of T3Es in Xam. Additionally our work highlights XopAO1 as a key T3E with role in virulence, suppression of PTI and ETI, and production of HR in Nicotiana tabacum. This could be an important target for engineering resistance in cassava against bacterial blight.

39

Session IV: Xop Effector Biology

O20-IV

The Role of the Environment in Host Specificity, Pathogen Virulence and Disease Spread

Mutka, A., Fentress, S., Vijayaraghavan, A., Wilson, M., Burke, J., Zimmerman, A., Bart, R.

Donald Danforth Plant Science Center Crop losses lead to food insecurity, especially in poorer communities and in the developing world. Xanthomonads are plant-associated bacteria that cause disease on most important crops. It is widely recognized that the host, the pathogen and the environment all contribute to disease severity. The genetic background of both the host and the pathogen can dramatically affect the outcome of a given interaction. Type three effectors, for example, are a group of bacterial virulence proteins whose presence can promote susceptibility or trigger strong resistance depending on the genetic background of the host. In the absence of a cognate plant resistance protein, most type three effectors contribute to susceptibility in a more subtle manner and are consequently difficult to study. Similarly, the influence of the environment over disease severity is less clear. We are combining traditional gene knockout strategies with a comparative genomics approach to further understand the role of type three effectors and the environment in Xanthomonas biology. Several exciting new phenotyping techniques are aiding our studies of host-pathogen interactions and will be discussed.

40

Session IV: Xop Effector Biology

O21-IV

Identification and Functional Analysis of Novel Xanthomonas euvesicatoria Type III Effectors

Doron Teper1, Georgy Popov1, Eran Bosis1, David Burstein2, Dor Salomon1, Tal Pupko2 and Guido Sessa1.

Dept. of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel Aviv, Israel. 2 Dept. of Cell Research and Immunology, Tel Aviv University, Tel Aviv, Israel. 1

The Gram-negative bacterium Xanthomonas euvesicatoria (Xcv) is the causal agent of bacterial spot disease in pepper and tomato. Xcv pathogenicity depends on a type III secretion (T3S) system that delivers effector proteins into host cells to suppress plant immunity and promote disease. The pool of known Xcv effectors includes about 30 proteins, most of them identified in the 85-10 strain. To identify additional Xcv 85-10 effectors, we applied a genome-wide machine learning approach, in which all ORFs were scored according to their propensity to encode effectors. Scoring was based on a large set of features including genomic organization, taxonomical dispersion, hrpdependent transcript accumulation, 5’ regulatory sequences, amino acid bias, and GC content. Thirty-six predicted effectors were tested for translocation into plant cells. Seven proteins harbored a functional translocation signal and their translocation relied on the HrpF translocon indicating that they are bona fide T3S effectors. Remarkably, four of them belong to yet undefined effector families. Inactivation of XopAP, a putative lipase, reduced the severity of disease symptoms in infected plants as reflected by a decrease in cell death and chlorophyll content in pepper leaves inoculated with the xopAP mutant as compared to the wild-type. In addition, XopAU was found to encode a biochemically active serine/threonine protein kinase that alters signaling of plant immunity by manipulating MAP kinase cascades.

41

Session V: TAL Effector Biology

O22-V Keynote talk

Moderately High-Throughput Xanthomonas Genome Sequencing for Population-Level TAL Effector Analysis

Adam J. Bogdanove1, Nicholas J. Booher1, Katherine E. Wilkins1, Sara C. D. Carpenter1, Li Wang1, Robert P. Sebra2, Steven L. Salzberg3, and Jan E. Leach4

Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY 14853 USA. 2 Icahn Institute for Genomics and Multiscale Biology and Department of Genetics & Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029 USA. 3 Departments of Biomedical Engineering, Computer Science, and Biostatistics and Center for Computational Biology, Johns Hopkins University, Baltimore, MD 21205 USA. 4 Bioagricultural Sciences and Pest Management, Colorado State University, Ft. Collins, CO 80523 USA 1

Pathogen-injected activators of host genes, TAL effectors play determinative roles in plant diseases caused by Xanthomonas spp. They are also important DNA targeting reagents. However, the structural repeats that determine their target specificity make TAL effector (tal) genes nearly impossible to assemble using next-generation, short reads. Long-read, single molecule, real-time sequencing (Pacific Biosciences) solves this problem. Using long-read data and an ensemble approach that first generates local, tal gene assemblies, we completely assembled de novo the genomes of one strain each of X. oryzae pv. oryzae and X. oryzae pv. oryzicola (Xoc) that we sequenced previously using the Sanger method, and corrected important errors in those references. Sequencing one more strain each revealed dynamic genome structure and striking plasticity in tal genes. Next, we completely sequenced 10 diverse Xoc strains, and, using RNA-seq, determined rice transcriptional responses to each. This allowed inference of important TAL effectors based on conservation, and of apparent convergent function based on shared predicted targets of sequence-distinct TAL effectors. We also observed conserved structural variants. Overall, with some exceptions, tal genes across strains largely reflect relationships based on housekeeping genes, suggesting predominantly vertical transmission. Of the roughly two dozen TAL effectors in each strain however, only five are strictly conserved. Applying a machine learning filter to better predict functional binding sites, we generated testable lists of candidate targets for these five. Our results pave the way for population-level studies to probe TAL effector evolution, inform development of durable resistance, and improve TAL effector utility.

42

Session V: TAL Effector Biology

O23-V

QueTAL: A Suite of Tools to Classify and Compare TAL Effectors Functionally and Phylogenetically

Alvaro L. Pérez-Quintero1, Léo Lamy1, Jonathan Gordon2, Aline Escalon2, Sébastien Cunnac1, Boris Szurek1, Lionel Gagnevin1 1 2

UMR IPME, IRD-CIRAD-Université Montpellier, Montpellier, France UMR PVBMT, CIRAD-Université de la Réunion, Saint-Pierre, France

Transcription Activator-Like (TAL) effectors from plant pathogenic bacteria can bind to the promoter region of plant genes and induce their expression. DNA-binding specificity is governed by a central domain made of nearly identical repeats, each determining the recognition of one base pair via two amino-acid residues (repeat variable diresidues or RVD). Knowing how TAL effectors differ from each other within and between strains would be useful to infer functional and evolutionary relations, but their repetitive nature precludes reliable use of traditional alignment methods. The suite QueTAL was then developed to offer new tools for comparison of TAL genes. The program DisTAL considers each repeat as a unit, transforms a TAL sequence into a sequence of coded repeats and makes pair-wise alignments between these coded sequences to construct trees. The program FuncTAL is aimed at finding TALs with similar DNA-binding capabilities. It calculates correlations between positional weight matrices obtained from the RVD sequence, and builds trees based on these correlations. The programs accurately represented phylogenetic and functional relations between TAL effectors using either simulated or reported data. When using the programs on a large set of TAL effector sequences, the DisTAL tree largely reflected the expected phylogeny. In contrast, FuncTAL showed that TALs with similar binding capabilities can be found between phylogenetically distant taxa. This suite will help users to rapidly analyse any TAL genes of interest and compare them to other available TAL genes and will hopefully help our understanding of TAL effector evolution. It is available at http://bioinfo-web.mpl.ird.fr/cgi-bin2/quetal/ quetal.cgi.

43

Session V: TAL Effector Biology

O24-V

Which Effectors in the TAL Arsenal of Xanthomonas citri pv. citri are Responsible for Host Specificity on Citrus?

Aline Escalon1, Laëtitia Guardini1, Stéphanie Javegny1, Boris Szurek2, Alvaro L. Pérez-Quintero2, Jonathan Gordon1, Pierre Lefeuvre1, Christian Vernière1, Olivier Pruvost1, Laurent D. Noël3, Matthieu Arlat4, Lionel Gagnevin1

UMR PVBMT (CIRAD/Université de la Réunion), F-97410 Saint-Pierre, La Réunion, France 2 UMR IPME (IRD/CIRAD/Université de Montpellier), F-34394 Montpellier, France 3 Laboratoire des interactions plantes micro-organismes (LIPM) UMR CNRSINRA 2594/441 F-31326 Castanet-Tolosan, France 4 Université Paul Sabatier, F-31062 Toulouse, France. 1

Transcription Activator Like (TAL) effectors are important factors for the pathogenicity of Xanthomonas. They are responsible for disease in susceptible plants by activating specific susceptibility genes, but they trigger defense responses in plants where this function has been hijacked to activate resistance genes. We analyzed the diversity of the arsenal of TAL genes in a large collection of Xanthomonas citri pv. citri corresponding to broad host range strains (pathotype A) and narrow host range strains (pathotype A* and AW) in order to investigate the role of TAL effectors on the definition of host range, as well as possible mechanisms of TAL evolution. We demonstrated that homologues of pthA4 are not only responsible for symptom formation but also drive the host specificity of X. c. pv. citri pathotypes. In contrast, additional TAL genes play a minor role and may constitute traces of acquisition and recombination, as well as a reservoir for future new TAL combinations. Comparisons of the potential Citrus target genes for the different TALs in X. c. pv. citri were performed in order to decipher the biological mechanisms of host specificity at the species scale.

44

Session V: TAL Effector Biology

O25-V

Transcriptional Activator-Like Effectors TAL6 And TAL11a Of Xanthomonas oryzae pv. oryzicola Suppress AvrXa7-Xa7 Mediated Defense In Rice

Zhi-Yuan Ji1,2†, Lu-Lu Cai1,2†, Wen-Xiu Ma1,2, Li Xiong1,2, Liang Liu1, Muhammad Zakria1,2, Guang-Hai Ji3, Li-Fang Zou1, and Gong-You Chen1,2 1 School of Agriculture and Biology, Shanghai Jiao Tong University/Key Laboratory of Urban (South) by Ministry of Agriculture, Shanghai, 200240, China. 2 State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. 3 Key Laboratory for Plant Pathology of Yunnan Province, College of Plant Protection, Yunnan Agricultural University, Kunming, 650201, China * Corresponding author, Gong-You Chen; Telephone: +86-021-3420-5873; Fax: +86-021-3420-5873; E-mail: [email protected]

The closely-related plant pathogens Xanthomonas oryzae pv. oryzicola and X. oryzae pv. oryzae cause bacterial leaf streak (BLS) and bacterial leaf blight (BLB) in rice, respectively. Unlike X. oryzae pv. oryzae, endogenous avirulence-resistance (avr-R) gene interactions have not been identified in X. oryzae pv. oryzicola-rice pathosystem, though both X. oryzae pv. oryzicola and X. oryzae pv. oryzae possess transcription activator-like effectors (TALEs), which are known to modulate R or S genes in rice. In this report, avrXa7, avrXa10 and avrXa27 from X. oryzae pv. oryzae were transferred into YNB017 and RS105, a hypovirulent strain and a hypervirulent one of X. oryzae pv. oryzicola, respectively. When YNB0-17 containing avrXa7, avrXa10 or avrXa27 was inoculated to rice, hypersensitive responses (HRs) were elicited in rice cultivars containing the R genes Xa7, Xa10 and Xa27, respectively. By contrast, RS105 expressing avrXa27 elicited an HR in a rice cultivar containing Xa27, but the expression of avrXa7 and avrXa10 in RS105 did not result in HRs in rice cultivars containing Xa7 and Xa10, correspondingly. Southern blot analysis demonstrated that YNB0-17 possesses only about 9 putative TALE genes, whereas the hypervirulent RS105 contains at least 20. Although YNB0-17 contains an intact type III secretion system (T3SS), its genome is lacking the T3SS effector genes avrRxo1 and xopO, which are present in RS105. The introduction of avrRxo1 and xopO into YNB0-17 did not suppress avrXa7- or avrXa10-triggered immunity in rice. Howerver, the transference of individual tale genes from RS105 into YNB0-17 led to the identification of tal6 that suppressed avrXa7-Xa7 mediated defense. Thus, YNB0-17 may be a useful recipient for discovering such the suppressors. This is the first report that co-evolutionally generated tale genes in X. oryzae pv. oryzicola suppress gene-for-gene defense against BLB, which may explain the lack of BLS resistant cultivars.

45

Session V: TAL Effector Biology

O26-V

Xanthomonas translucens – A Role of Type III Effectors in Pathogenicity

Céline Pesce1,2, Edwige Berthelot1, Daniel Garcia-Seca1, Claude Bragard2, Ralf Koebnik1

UMR Interactions Plantes Microorganismes Environnement, IRD-Cirad-UM, Montpellier, France 2 Earth and Life Institute, Applied Microbiology Phytopathology, Université catholique de Louvain, Belgium 1

Xanthomonas translucens (Xt) is the causal agent of Bacterial Leaf Streak (BLS), the most common bacterial disease of small grain cereals such as wheat and barley. This disease has been reported at diverse locations worldwide until the end of last century and received increased attention in recent years. BLS of barley and wheat is a seed-borne disease and thus a constraint for international germplasm exchange. Several countries list Xt as a quarantine organism. Yield losses as high as 40 percent have occurred in the most severely diseased fields in United States, although losses are generally 10 percent or less. To cause disease, most xanthomonads depend on a highly conserved type III secretion system (Hrp system), which translocates type III effectors into plant host cells. In order to identify new pathogenicity factors, we sequenced the genomes of five Xt strains. Comparative genomics revealed specific features for the Xt Hrp system and the type III effector repertoire. New candidate type III effectors were studied by reporter gene fusions. Mutants in the Hrp system and in predicted effector genes were generated. Pathogenicity assays on barley revealed a substantial contribution of the Hrp system and of one distinct Transcription Activator-Like (TAL) effector to pathogenicity. A set of tal genes was sequenced in order to identify new susceptibility genes in barley. Most notably, we identified a new gene that is essential for virulence of Xt. Latest results will be presented and new insight into the molecular mechanism of the Xt -cereals interaction will be discussed.

46

Session VI: Other Pathogenicity Factors

O27-VI Keynote talk

Construction and Analysis of a Transcriptional Regulatory Network of Main Pathogenicity-Regulatory Genes in Xanthomonas campestris pathovar campestris

Wei Jiang, Wei Liu, Guigang Xie, Xiangna Niu, Bo-Le Jiang, Guang-Tao Lu, Dong-Jie Tang, Yong-Qiang He*, Ji-Liang Tang*

State Key Laboratory for Conservation and Utilization of Subtropical Agrobioresources; College of Life Science and Technology, Guangxi University, Nanning, 530004, China *Corresponding authors: Ji-Liang Tang ([email protected]); Yong-Qiang He ([email protected]) Xanthomonas campestris pathovar campestris (Xcc), the causal agent of black rot disease of crucifers, is a model bacterium for studying molecular microbeplant interactions. Previously, we found that mutation in any of the nine regulatory genes (clp, colR, hpaR, hpaR1, hrpG, hrpX, vemR, rsmA and zur) of Xcc 8004 resulted in significant reduction in virulence on the host plant Chinese radish and in hypersensitive response on the non-host plant pepper ECW-10R, thus defined as pathogenicity-regulatory genes of Xcc. In this work, a transcriptional regulatory network of the pathogenicity-regulatory genes was constructed by hierarchical clustering analyses of their regulons generated by transcriptome RNA-Seq. The regulatory network visualized by Pajek showed intuitively the interrelations among the pathogenicity-regulatory genes and their co-regulated genes. hrpX, the core node, is positively regulated by clp, hrpG, rsmA and zur and positively regulates hpaR, hrp operons (hrpA to hrpF) and many effector genes. vemR also positively regulates hrp operons, but there is no transcriptional regulation between vemR and hrpX. colR can only positively regulate hrpC operon and hpaR positively regulate hrpD operon. We also verified that hpaR1 negatively regulates hrpG in vitro, but induce hrpG expression in planta. Furthermore, in addition to regulating putative pathogenicity genes, these pathogenicity-regulatory genes also control the expression of genes with different functional categories. It is worth to verify whether these co-regulated genes play a role in bacterial pathogenicity. Our results re-constructed a complex regulatory network for regulating virulence in Xcc, providing a valuable reference for elucidating the molecular pathogenesis of phytopathogenic bacteria.

47

Session VI: Other Pathogenicity Factors

O28-VI

Searching for Determinants of Host Specificity in X. axonopodis pv. phaseoli and X. fuscans subsp. fuscans

Laurana Serres-Giardi1*, Nicolas W.G. Chen1,2*, Martial Briand1, Mylene Ruh1,2, Sophie Bonneau1, Armelle Darrasse1, Lionel Gagnevin3, Ralf Koebnik4, Laurent D. Noël5, Marie-Agnes Jacques1

INRA, UMR1345 Institut de Recherche en Horticulture et Semences, SFR 4207 QuaSaV, Beaucouzé, France. 2 AgroCampus-Ouest, UMR1345 Institut de Recherche en Horticulture et Semences, SFR 4207 QuaSaV, Angers, France. 3CIRAD, UMR PVBMT, F-97410 Saint-Pierre, La Réunion, France. 4 IRD, UMR RPB, F-34394 Montpellier Cedex 5, France. 5 CNRS, LIPM UMR 2594, F-31326 Castanet-Tolosan, France. * Equal contribution 1

Phytopathogenic bacteria from the genus Xanthomonas can cause disease to more than 400 plant species. However, at the infrasubspecific level, each pathovar is able to infect only one or few plant species, indicating that host specificity is highly important within the genus Xanthomonas. Analyses of genes classically described as involved in pathogenicity (type III effectors, sensors, adhesins) indicate that for these genes, each Xanthomonas pathovar possesses a repertoire correlated to its host of isolation (Hajri et al, 2009; Mhedbi-Hajri et al, 2011). Common bacterial blight of bean is caused by polyphyletic strains coming from four genetic lineages of either X. axonopodis pv. phaseoli (Xap) or X. fuscans subsp. fuscans (Xff). These four genetic lineages belong to the X. axonopodis species, as described by Vauterin and colleagues in 1995, but these genetic lineages are more or less distant to each other, phylogenetically. Thus, their ability to infect common bean corresponds to a functional convergence that can be explained at the molecular level either by convergent evolution or by horizontal gene transfer. In order to search for putative genetic determinants of host specificity in Xap-Xff, we performed comparative genomics analyses on 75 genomes representative for the diversity of Xanthomonas. Theses analyses, based on the détection of i) gene repertoires and ii) nucleotidic patterns, led to a list of genes presenting specific patterns in Xap-Xff strains. In all, we present a global overview of the genes putatively involved in adaptation of Xap-Xff strains to common bean and discuss on how these genes were acquired by phylogenetically distant strains.

48

Session VI: Other Pathogenicity Factors

O29-VI

ATP-Dependent RNA Helicase HRPB is Involved in mRNA Regulation of Type IV Operon in Xanthomonas citri subsp. citri

L.M. Granato, M.O. Andrade, S.C. Picchi, M.A. Takita, A.A. de Souza, N. Wang, M.A. Machado

Biotechnology Laboratory, IAC – Centro de Citricultura - Sylvio Moreira, Cordeiropolis, Brazil Microbiology Laboratory, UF - Citrus Research and Educational Center, Lake Alfred, USA RNA helicases are enzymes that catalyze the separation of double-stranded RNA (dsRNA) using an ATP-dependent activity. DEAD/DEAH protein family participate many different aspects of RNA metabolism such as RNA synthesis, RNA folding, RNA-RNA interactions, RNA localization, and RNA degradation. Several bacterial DEAD/DEAH proteins have been studied extensively due to their importance in E. coli, Bacillus subtilis and Staphylococcus. Interestingly, in Staphylococcus aureus, the DEAD-box protein CshA was involved in biofilm formation via modulation of agr mRNA stability. In E. coli, it was demonstrated that a DEAH-box RNA helicase is involved in mRNA processing of a fimbrial operon. In this study, the relative expression of type IV pili genes between the wild type and mutant strains of Xanthomonas citri subsp. citri was determined by qRT-PCR. Effect of hrpB on fimA mRNA stability was determined by measuring the abundance of the fimA and gyrA (control) transcripts after the addition of 10 mg/mL ciprofloxacin. Mapping of the 5’ - UTR sequences of fimA (XAC3241) transcript was determined using the 3’/5’ RACE Kit (Roche). Our data show that hrpB is involved in the regulation of fimA (XAC3241). The putative ATP-dependent RNA helicase HrpB plays a role in alteration of fimA mRNA structure, which promotes stability of fimA RNA. It is to the best of our knowledge the first time that a DEAD-box RNA helicase is implicated in the regulation of type IV pili genes in Xanthomonas citri subsp. citri.

49

Session VI: Other Pathogenicity Factors

O30-VI

Full Elucidation of the Hitherto Unknown Structure of Albicidin, a Potent Antibiotic Produced by Xanthomonas albilineans

Stéphane Cociancich1*, Alexander Pesic2, Daniel Petras2, Stefanie Uhlmann2, Julian Kretz2, Vivien Schubert2, Laura Vieweg2, Sandrine Duplan1, Mélanie Marguerettaz1, Julie Noëll1, Isabelle Pieretti1, Manuela Hügelland2, Sebastian Kemper2, Andi Mainz2, Philippe Rott1,3, Monique Royer1 and Roderich D. Süssmuth2

Cirad, UMR BGPI, F-34398 Montpellier cedex 5, France Institut für Chemie, Technische Universität Berlin, D-10623 Berlin, Germany 3 Present address: University of Florida, IFAS, Everglades Research & Education Center, Belle Glade, FL, USA *Contact: [email protected] 1 2

Albicidin is a potent DNA gyrase inhibitor produced by the sugarcane pathogenic bacterium Xanthomonas albilineans. As such, this molecule blocks the differentiation of chloroplasts, resulting in appearance of narrow white stripes on sugarcane leaves that are characteristic of leaf scald disease. Albicidin targets the bacterial gyrase by a mechanism that is different from the one of other DNA gyrases inhibitors like coumarins or quinolones. It also exhibits antibacterial activity at nanomolar concentrations against Escherichia coli and to a lower extent against numerous Gram-negative and -positive human pathogenic bacteria. A decade of intense work was necessary to decipher albicidin’s biosynthetic pathway and to elucidate its astonishing never-seen-before structure. Albicidin is produced by a hybrid PKS/NRPS (polyketide synthase/non ribosomal peptide synthetase) system. Such ribosome-independent systems consist of modular megasynthetases which operate in an assembly-line fashion to activate, modify and link mostly unusual aminoacid building blocks, finally resulting in complex bioactive peptide-like molecules. The structure of albicidin, which was predicted by former in silico sequence analyses of its PKS/NRPS gene cluster, was ascertained by means of mass spectrometry and nuclear magnetic resonance spectroscopy. We were able to demonstrate that albicidin exhibits a linear polyaromatic penta-peptidic structure containing the rare aminoacids para-aminobenzoate and cyanoalanine. The determination of the structure of albicidin allowed the development of a protocol for the chemical synthesis of this complex molecule. Consequently, new research, such as structure-activity relationship studies, will now be possible. New insights into biosynthesis pathway and structural determination for albicidin will be presented.

50

Session VII: Plant Defense Activation and Suppression

O31-VII Keynote talk

Tomato 14-3-3 Proteins: Signaling Components of Immunity and Common Targets of Xanthomonas Effectors

Zoë Dubrow1, Sunitha Sukumaran2, Jung-Gun Kim1, Guido Sessa2, and Mary Beth Mudgett1

Department of Biology, Stanford University, Stanford, CA, USA 94305 Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Tel Aviv, Israel 6997801

1 2

The goal of this research is to identify 14-3-3 proteins that are involved in plant immunity and to determine how bacterial pathogens employ the type III secretion (T3S) system and its cognate effector proteins to manipulate the function of 14-3-3s. Mounting evidence indicates that 14-3-3s, a class of eukaryotic phospho-binding proteins with scaffolding activity, play central roles in the assembly and disassembly of eukaryotic signaling complexes, ultimately controlling the kinetics and magnitude of cellular responses to given stimuli. Recent work suggests that 14-3-3s are required for proper execution of plant immune responses during infection. Specific 14-3-3s have been linked to regulation of pathogen-triggered immunity (PTI) and effector-triggered immunity (ETI). Furthermore, emerging data suggests that T3S effectors physically associate with plant and animal 14-3-3s, indicating that diverse pathogens have evolved mechanisms to subvert and/or co-opt 14-3-3 function to promote pathogenicity. Here we examined the roles of 11 tomato 14-3-3 isoforms in PTI and ETI. We also identified the suite of Xanthomonas euvesicatoria T3S effectors that target tomato 14-3-3s and characterized biochemical features impacting 14-3-3/effector complex formation. Our data reveal that multiple tomato 14-3-3 isoforms contribute to anti-Xanthomonas immunity and a small group of effectors interact with multiple 14-3-3 isoforms. These data and recent progress in understanding the impact of 14-3-3/effector complex formation on the proper execution of tomato immune responses will be discussed.

51

Session VII: Plant Defense Activation and Suppression

O32-VII

An Adaptor Kinase Confers Expanded Recognition Specificity to a Plant NLR

Brice Roux1,2#, Guoxun Wang3#, Feng Feng3#, Endrick Guy1,2, Lin Li4, Nannan Li4, Martine Lautier1,2,5, Marie-Françoise Jardinaud1,2, Matthieu Chabannes1,2,7, Matthieu Arlat1,2,5, She Chen4, Chaozu He6, Jian-Min Zhou3* and Laurent D. Noël1,2*

INRA, Laboratoire des Interactions Plantes Micro-organismes (LIPM), UMR 441, Castanet-Tolosan, France 2 CNRS, Laboratoire des Interactions PlantesMicro-organismes (LIPM), UMR 2594, Castanet-Tolosan, France 3 State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,No. 1 West Beichen Road, Beijing 100101, China 4 National Institute of Biological Sciences, Beijing 102206, China 5 Université Paul Sabatier, Toulouse, France 6 Hainan University, Haikou, China 7 Present address: CIRAD, UMR BGPI, F-34398 Montpellier Cedex 5, France # These authors contributed equally to the work. * [email protected]; [email protected] 1

Effector proteins of pathogenic microbes utilize diverse biochemical activities to perturb cellular processes in host plants and animals, promoting parasitism. However, these biochemical activities can betray the pathogen by triggering host immunity when plants and animals carry cognate NOD-Like Receptors (NLRs) as a result of host-pathogen co-evolution. The Xanthomonas campestris effector protein AvrAC/XopAC inhibits plant immunity by uridylylating the Arabidopsis BIK1 kinase. Here, we show that AvrAC also uridylylates a related kinase, PBL2, which is required for resistance but dispensable for AvrAC virulence function in plants. PBL2 acts as a BIK1 decoy and is perceived by a stable pre-activation complex made of the pseudokinase RKS1 of the ZRK family and the NLR protein ZAR1. ZAR1 and the ZRK ZED1 were previously reported to confer recognition of an unrelated Pseudomonas syringae effector. Our results thus show how a plant ZRK adaptor kinase specifies and expands the recognition spectrum of an evolutionary-conserved NLR to compete in the arms-race against multiple pathogens.

52

Session VIII: Novel Disease Resistance and Control

O33-VIII Keynote talk

TALEN- and CRISPR-Mediated Genetic Variations Lead to Resistance to Bacterial Blight in Rice

Bing Yang

Iowa State University, U.S.A. TAL (transcription activator-like) effectors in Xanthomonas bacteria underlie pathogenesis by transcriptionally activating host plant genes that are vulnerable to disease or confer resistance by binding to the promoters of target genes in a simple cipher, -i.e. one of the central near-identical 34 amino acid repeats of TAL effector recognizes one base of the target DNA sequence in a sequential order, and one particular type of repeats preferentially corresponds to one of four nucleotides. Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), represents an excellent model for understanding the TAL effector biology and genetically engineering disease resistance. Xoo depends on a group of TAL effectors to condition a state of disease susceptibility by targeting three members of SWEET gene family in rice. OsSWEET11 is targeted by PthXo1, OsSWEET13 by PthXo2, and OsSWEET14 by AvrXa7, PthXo3, TalC and Tal5. Targeting occurs with binding of individual TAL effectors to their cognate effector binding elements (EBEs) within the promoters of SWEETs. Fortunately, such virulence strategy presents the targets exploitable for engineering novel disease resistance. We have developed the TAL effector nuclease (TALEN) and CRISPR/Cas9 technologies in rice and successfully used them to precisely modify the EBEs within the promoters of the SWEET disease susceptibility (S) genes. Multiple TALENs are custom-engineered to precisely edit OsSWEET11 and OsSWEET14, while CRISPR/Cas9 has been used to target each or combination of all three SWEET S genes. The resultant promoter modifications in rice plants result in loss of inducibility of the disease genes and concomitantly loss of disease susceptibility (or gain of resistance) to pathogen. The TALEN or CRISPR/Cas9 gene constructs can be eliminated in some modified plants through genetic crosses. The results demonstrate the feasibility of using TALENs and CRISPR/ Cas9 for targeted editing of important genes for crop improvement and also raise the prospect of producing genetically modified plants without a trace of “foreign” DNA left in the genome.

53

Session VIII: Novel Disease Resistance and Control

O34-VIII

Novel Resistance Sources for Bacterial Blight and Bacterial Leaf Streak of Rice in West Africa

Bossa-Castro, A.M.1, Raghavan, C.2, Delorean, E. E.1, Vera-Cruz ,C.2, Leung, H.2, Mosquera, G.3, Verdier, V.1,4, Leach, J. E. 1

Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, USA 2 International Rice Research Institute (IRRI), Los Baños, Philippines 3 International Center for Tropical Agriculture (CIAT), Palmira, Colombia 4 Institut de Recherche pour le Développement IRD-CIRAD-UM2, Montpellier, France 1

Bacterial blight (BB) and bacterial leaf streak (BLS) are caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively. These devastating diseases of rice are widespread in Africa and Asia, causing considerable losses and yield reduction. In Africa, effective BB or BLS resistance is largely absent in the rice germplasm currently used, and no single resistance genes have been identified for control of African Xoo and Xoc strains. Our goal is to identify novel, broad-spectrum resistance sources to control BB and BLS in rice, using a new mapping resource, a Multi-parent Advanced Generation Inter-Cross (MAGIC) population. The advantage of MAGIC populations is the increased level of recombination and the precision and resolution to detect quantitative trait loci (QTL). At the fourth (S4) and eight (S8) selfed generations, subsets of single seed descent lines and founders were genotyped by sequencing. Both S4 and S8 subsets were phenotyped for resistance to Xoc and Xoo African strains. Genome-wide association and interval mapping analyses were performed to map disease resistance QTL. We identified QTL that potentially confer broad-spectrum resistance effective against African Xoo and Xoc strains. Because the MAGIC population was derived from elite cultivars, ultimate use of resistance sources in breeding programs will be expedited.

54

Session VIII: Novel Disease Resistance and Control

O35-VIII

Engineering Resistant Cassava (Manihot esculenta, Crantz) Plants Against Cassava Bacterial Blight

Paula Díaz-Tatis1, Juan Camilo Ochoa1, Mariana Herrera1, Adriana Bernal2, Paul Chavarriaga3 Camilo López1 * 1 Manihot Biotech, Departamento de Biología, Universidad Nacional de Colombia, Cra 30 # 45-03, Bogotá, Colombia. 2 Laboratory of Mycology and Plant Pathology, Universidad de los Andes, Cra 1 # 18A-10, Bogotá, Colombia 3 Centro Internacional de Agricultura Tropical, CIAT, Km 17 Recta Cali-Palmira, Colombia. *Corresponding autor: [email protected]

Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv manihotis (Xam), is a devastating disease in all regions where cassava is cultivated. To generate broad spectrum and durable CBB resistance it is necessary pyramiding several R genes. We have identified two candidate resistance genes, named RXam1 and RXam2 which colocalize with QTLs associated to resistance against Xam. RXam1 codes for a RLK (Receptor-Like Kinase) protein. We generated several transgenic cassava lines overexpressing RXam1. In vitro plants of three lines showed reduced symptoms and reduced bacterial growth after Xam infection compared to empty vector transgenic plants. The second cassava gene, RXam2, codes for a NB-LRR protein. Using RNAi we generated RXam2-silenced transgenic plants, which were more susceptible to several Xam strains than non-transformed plants. These data suggest that RXam2 is a broad-spectrum resistance gene against Xam. In addition, an autoactive version mutated in the MHD motif (NB domain) of RXam2 was generated through site directed mutagenesis and was able to generate a Hypersensitive Response (HR) by transient agroinfiltration in cassava and tobacco leaves. The autoactive version of RXam2 was cloned under a TALE1Xam-inducible promoter and transient expression in tobacco showed a strong HR when coinfiltrated with a plasmid containing the TALE1Xam gene. Several independent transgenic stable lines are being evaluated to assess TALE1Xam inducibility. Finally, several cassava transgenic plants overexpressing Bs2 from pepper were obtained and showed constitutive, typical immune responses. The introduction of these genes by traditional or non-conventional breeding strategies may generate durable and broad-spectrum CBB resistance.

55

Session VIII: Novel Disease Resistance and Control

O36-VIII

Informed Discovery of xa40(T), a Novel OsSWEET14 Allele from Wild Rice Conferring Broad-Spectrum Resistance to Bacterial Blight of Rice

Mathilde Hutin1, Francois Sabot2, Alain Ghesquière2, Ralf Koebnik1, Boris Szurek1

Interactions Plantes Microorganismes Environnement, IRD, Montpellier, France 2 Diversité, Adaptation et Développement des Plantes, IRD, Montpellier, France 1

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating bacterial disease of rice, a crop that feeds more than half of the world’s population. Xoo virulence critically depends on the TAL (Transcription Activator-Like) effector-dependent activation of members of the SWEET family of sugar transporter genes, presumably resulting in redirection of sugars into the xylem as a carbon source for bacterial colonization. One SWEET gene in particular, OsSWEET14, functions broadly as a major bacterial blight susceptibility gene, which is targeted by numerous Xoo strains. We sequenced the OsSWEET14 promoter region of 169 accessions of wild and cultivated Oryza species, and uncovered an allele with a deletion of 18-bp overlapping with the binding sites targeted by three out of four TAL effectors known to activate the gene. We show that this allele, which we call xa40(t), confers resistance against 50% of the tested Xoo strains representative of various geographic origins and genetic lineages. xa40(t) is conserved in all 30 tested Oryza glaberrima accessions, as well as in most accessions of its wild ancestor O. barthii. xa40(t) is the first plant disease resistance gene identified through a knowledge-based molecular screen. It illustrates the potential of exploiting naturally-occurring variation in disease-susceptibility genes to dispossess the pathogen of a host factor required for proliferation, thus providing a novel paradigm for accelerated genomics-based crop improvement.

56

Session VIII: Novel Disease Resistance and Control

O37-VIII

A Rice Blight Resistance Gene Confers Resistance Against BLB and BLS

Hutin Mathilde1, Cesari Stella2, Chalvon Veronique2, Tuan Tran Tu1, Szurek Boris1, Kroj Thomas2

UMR Interactions Plantes Microorganismes Environnement, IRD-Cirad-UM, Montpellier, France 2 Biologie et Génétique des Interactions Plante-Parasite, INRA, Montpellier, France. 1

Avirulence (AVR) proteins are recognized by resistance (R) proteins in a direct or indirect manner. The avirulence protein AVR1-CO39 from the rice blast pathogen Magnaphorte oryzae is recognized by its cognate R protein RGA5 from rice upon direct binding. This recognition activates disease-resistance signaling pathways leading to the inhibition of pathogen growth and the induction of a localized programmed cell death called the hypersensitive response (HR). Avirulence (AVR) proteins are recognized by resistance (R) proteins in a direct or indirect manner. RGA4 and RGA5 (PICO39) are two NB-LRR protein-coding genes required for the recognition of the Magnaporthe oryzae effector AVR1CO39. AVR1-CO39 is recognized by RGA5 and this interaction suppresses the inhibition of RGA4 by RGA5, thus activating disease-resistance signaling pathways leading to the inhibition of pathogen growth and the induction of a localized programmed cell death (HR). Here we assessed the potential of using the AVR1-CO39/PI-CO39 interaction to confer resistance against Bacterial Leaf Blight (BLB) and Bacterial Leaf Streak (BLS) caused by the vascular pathogen Xanthomonas oryzae pv. oryzae (Xoo) and the non-vascular Xanthomonas oryzae pv. oryzicola (Xoc), respectively. Transgenic rice lines carrying Avr1-Co39 cloned downstream of a TAL effector-responsive promoter were generated in two varieties of rice containing Pi-Co39 and pi-Co39, respectively. These lines were challenged with virulent Xoo and Xoc strains carrying or not the artificial TAL effectors designed to induce specifically Avr1-Co39. Our results show that activation of the AVR1-CO39/PI-CO39 interaction is able to confer resistance against both pathovars of X. oryzae. Furthermore, a similar strategy was employed to activate RGA4, showing that its overexpression is sufficient to activate disease-resistance signaling pathways. Overall our results highlight the potential of exploiting rice blast resistance genes to control bacterial diseases caused by Xanthomonas species.

57

Session I: Diversity and Diagnosis

P1-I

Characterization of Cassava Bacterial Blight in Venezuela: Diversity of Xanthomonas axonopodis pv. manihotis (Xam) and its Talome

Carolina Flores1, Leidy Rache2, Camilo Lopez3, Ralf Koebnik1, Valérie Verdier1, Adriana Bernal2, Boris Szurek1

UMR Interactions Plantes Microorganismes Environnement, IRD-Cirad-UM, Montpellier, France 2 Laboratorio de Micología y Fitopatología, Universidad de Los Andes, Bogotá D.C., Colombia 3 Universidad Nacional de Colombia, Bogotá D.C., Colombia 1

Cassava Bacterial Blight (CBB) which is caused by Xanthomonas axonopodis pv. manihotis (Xam) is considered the most important bacterial disease threatening cassava production in Venezuela, with greater impact in eastern and savanna regions. CBB was first reported in Venezuela in the 70’s. The genetic diversity and variation in virulence of Xam was analyzed on field samples collected in the late 90’s. Since then no information as to the evolution and actual structure of Xam population is available. To fill this gap, a field survey of CBB will be performed from 2015 to 2017 in different ecozones where cassava is cultivated in Venezuela. Upon processing of symptomatic leaf samples, a new collection of Xam strains will be established. Diversity analysis which will be conducted through a Multiple Locus VNTR Analysis (MLVA) scheme, which is currently the most advanced tool for fast, highthroughput and high-resolution infraspecific molecular typing of bacterial isolates. For the most representative haplotypes, pathogenicity assays will be performed on a reference set of 10 cassava varieties and the diversity of their Transcription Activator-Like (TAL) effectors repertoire will be assessed. This knowledge will be key to discover new pathogen-informed sources of cassava resistance to control CBB in Venezuela.

58

Session I: Diversity and Diagnosis

P2-I

Toxin-Antitoxin Distribution and Adaptive Aspects on Xanthomonas Genomes: Focus on Xanthomonas citri

Martins, PMM; Silva NV; Takita, MA; de Souza, AA Centro APTA Citros Sylvio Moreira/IAC, Cordeirópolis, São Paulo, Brazil Prokaryotic toxin-antitoxin operons (TA) were first described as systems designed to prevent plasmid loss in bacteria. Later, studies showed that other biological functions could be assigned to them, like stress response and persister cell formation. However, few studies have analyzed the TA systems on the genomes of phytopathogens, and their possible roles in helping bacteria to survive to different environmental stresses. This may be especially important to pathogens like Xanthomonas spp that are supposed to thrive epiphytically before host infection. In this study, we analyzed 10 Xanthomonas genomes, assessing the nature and number of TA systems found in each one. No genome seemed to harbor RelB and HicA, and the most common toxin domain was VapC. Only one TA system was found exclusively in every Xanthomonas, suggesting a specific role on adaptive aspects regarding its environment and host interaction. Besides, some TAs appeared in a strain-specific manner, like HigBA, CcdAB and HipAB. We found that citrus-infecting pathovars have, on average, 3 times more TAs than any other Xanthomonas spp here studied, outnumbering by far X. albilineans, the strain that has the lowest number of TAs. This fact may be related to its specific host, since citrus is a perennial crop, therefore rendering X. citri exposed to more environmental challenges over the years. This study shows that Xanthomonas spp have quantitative and qualitative differences on TA systems, which may have implications on its survival abilities in each environment.

59

Session I: Diversity and Diagnosis

P3-I

Whole-Genome Sequencing of 18 Strains Reveals High Polymorphism in the Lipopolysaccharides Locus of Xanthomonas albilineans

Isabelle Pieretti1, Stéphane Cociancich1, Sébastien Carrère2, Alexandre Morisset1, Philippe Rott13 and Monique Royer1

CIRAD UMR BGPI, TA A-54/K, Campus international de Baillarguet, F-34398 Montpellier Cedex 5, France 2 INRA, UMR LIPM, F-31320 Castanet-Tolosan, France 3 University of Florida, IFAS, Belle Glade FL 33430, USA. 1

Xanthomonas albilineans causes leaf scald disease of sugarcane and exhibits distinctive pathogenic mechanisms, ecology and taxonomy when compared to other species of Xanthomonas. For example, X. albilineans does not possess the xanthan gum biosynthesis gene cluster and the Hypersensitive response and pathogenicity type 3 secretion system (Hrp-T3SS). X. albilineans also exhibits large intra-species variability as illustrated with occurrence of three serotypes and 10 PFGE genetic groups revealed by serotyping and PulsedField Gel Electrophoresis (PFGE), respectively. In this study, we performed a multi-locus sequence analysis using 15 strains spanning the PFGE diversity of X. albilineans, three strains of Xanthomonas sacchari and two strains belonging to the tentatively novel species X. pseudoalbilineans. Analysis of the lipopolysaccharides (LPS) genes located between the conserved etfA and metB genes revealed three different allelic forms of the LPS locus named LPS1, LPS2 and LPS3, associated to serotype 1, serotype 2 and serotype 3 of X. albilineans, respectively. Locus LPS1 contains five specific genes and shares seven genes with locus LPS3. Locus LPS2 contains 10 specific genes and shares five genes with locus LPS3. The 14 genes of locus LPS3 are conserved in two strains of X. sacchari. Strains belonging to a given PFGE group belong to the same serotype or exhibit the same LPS allelic form, suggesting that the gene transfer events that resulted to the distribution of the three LPS allelic forms in X. albilineans occurred before the differentiation of the PFGE lineages of this sugarcane pathogen.

60

Session I: Diversity and Diagnosis

P4-I

Analysis of the Population Diversity of Xanthomonas axonopodis pv. manihotis (Xam) in Different Regions of Colombia Using VNTRS

Leidy Rache1, Ralf Koebnik2, Adriana Bernal1

Laboratorio de Micología y Fitopatología, Universidad de Los Andes, Bogotá D.C., Colombia 2 UMR Interactions Plantes Microorganismes Environnement, IRD-Cirad-UM, Montpellier, France 1

Studies of genetic variation of phytopathogenic bacteria are not only important to understand epidemiology of diseases produced by these microorganisms but also to propose strategies of disease control. Therefore, since 1990 there have been several studies of population genetic diversity of Xanthomonas axonopodis pv. manihotis (Xam) in Colombia. Xam is the causal agent of cassava bacterial blight, and its infection highly reduces crop yield. Previous studies have reported that Xam has a high population diversity that rapidly changes over time. Consequently, varietal resistance may be lost, and new approaches to control this disease are necessary. Furthermore, these studies show the importance of continuous analysis of population dynamics. The objective of this research is to use Multiple-Locus Variable number tandem repeat Analysis (MLVA) to genotypify Xam populations and analyze their dynamics over time. In addition, we will analyze if special features, such as the high diversity previously found in Chinú, is still present over time. To develop this study, strains of Xam have been collected from different regions of Colombia since 2013 and will be collected until 2016. The isolates are being analyzed using primers to amplify four minisatellite loci and eighteen microsatellite loci. The results from this project will improve the knowledge of the current disease status in the country. It will also allow us to propose alternatives for disease control in the field and to compare the genetic diversity found in Colombia with Xam strains isolated from other countries in the region and around the world.

61

Session I: Diversity and Diagnosis

P5-I

Multilocus Sequence Analysis Reveals High Diversity Among Xanthomonas Strains Affecting Tomato Crops in Uruguay.

Siri M.I.1, Lapaz M.I.1, Hernández F.1, Montelongo M.J.2, Maeso D.3, and Pianzzola M.J.1

Laboratorio de Microbiología Molecular, Cátedra de Microbiología, Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo, Uruguay. 2 Cátedra de Fitopatología, Departamento de Protección Vegetal, Facultad de Agronomía, Universidad de la República, Montevideo, Uruguay. 3 Instituto Nacional de Investigación Agropecuaria, INIA Las Brujas, Canelones, Uruguay. 1

Bacterial spot is an important disease of tomato (Solanum lycopersicum) in both tropical and more temperate regions. In Uruguay is considered a major constraint to tomato production causing extensive damage and significant economic losses to local farmers. The disease is caused by phenotypically and genetically heterogeneous strains of the genus Xanthomonas actually assigned to four species: X. euvesicatoria, X. perforans, X. gardneri, and X. vesicatoria. In this work we examined the genetic diversity of a collection of 35 Xanthomonas strains isolated from tomato crops in different locations in Uruguay as part of a national research program to update inventories of agricultural pathogens. To identify the isolates at the species level we applied a Multilocus Sequence Analysis (MLSA) scheme based on five housekeeping genes (atpD, dnaK, efp, gyrB and rpoD). This approach allows us to determine that X. vesicatoria and X. gardneri were the species recovered prevalently from tomato crops in Uruguay. The analysis revealed the existence of several haplotypes within each species, being X. vesicatoria the more diverse. This study provides the first ever comprehensive description of the status of Xanthomonas species that cause bacterial spot of tomato in Uruguay.

62

Session II: Epidemiology and Ecology

P6-II

Phylogeography and Population Structure of Xanthomonas fragariae to Identify Sources and Pathways of Bacterium Through Plant Material Trade

Michael Gétaz1, Joël F. Pothier1, Marjon Krijger2, Jan van der Wolf2, Brion Duffy1

Zürich University of Applied Sciences ZHAW, Institute of Natural Resource Sciences, Environmental Genomics and Systems Biology research group, Wädenswil, Switzerland. 2 Wageningen UR, Biointeraction and Plant Health, Wageningen, the Netherlands 1

Angular leaf spots on strawberry are caused by Xanthomonas fragariae with particular severe effects under protected cultivation with high density plots aided by high humidity and sprinkler irrigation systems. X. fragariae was first observed in the USA in 1960, in Europe in 1970 and then spread worldwide in strawberry growing regions. Its quick spread is thought to be due to importation of plant material through trade and more generally of human activities. Symptomless infected strawberry and latent invisible infections were additional factors influencing spread through plant material. In addition to quick, reliable detection and identification of this pathogen, there is a real need for reliable methods accurately discriminating between strains for crop surveillance, outbreak investigation and establishing disease control strategies. As relatively low diversity was observed among X. fragariae strains of various geographic and time origins, the discrimination power of several molecular markers was determined. Among these markers for source tracking purpose, variable number of tandem repeats (VNTRs) would be an efficient genotyping method where numbers of repeats would act as molecular clocks with sufficient resolution. Clustered regularly interspaced short palindromic repeats (CRISPRs) would also constitute useful markers in epidemiology and host-bacteria surveys bringing complementary information for genetic diversity characterization. In the frame of the EU DROPSA project, we aim to apply strain-level molecular markers in order to get better understanding on population structures of the quarantine pathogen X. fragariae and how the disease can emerge and spread over a given geographical region.

63

Session III: Pathogenesis and Regulation

P7-III

Study of XacPNP in Xanthomonas citri subsp. citri

María Victoria Barcarolo, Betiana S. Garavaglia, Florencia A. Ficarra, Natalia Gottig, Jorgelina Ottado

Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET-UNR) Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker. This phytopathogen contains a gene encoding a plant natriuretic peptide (PNP)-like protein (XacPNP) that shares significant sequence similarity and identical domain organization with PNPs and it is not present in any other bacteria. PNPs are peptide hormones involved in regulation of ions and water homeostasis. We have previously shown that XacPNP is involved in plant-pathogen interaction modulating host homeostasis to the benefit of the pathogen, such as the improvement of host photosynthesis, rendering a healthier tissue that favors the survival of this biotrophic bacterium. However, a possible role of XacPNP on bacterial physiology has never been studied. XacPNP is synthesized as a preprotein and probably processed during secretion. We found that expression of the recombinant XacPNP precursor caused bacterial lysis in Esherichia coli, and this effect was not observed when the predicted mature protein was expressed. Moreover, the expression of XacPNP precursor in Xcc, and in a variant of Xanthomonas axonopodis pv. vesicatoria, impaired bacterial survival of both strains under saline stress conditions. These results suggest a possible role of XacPNP in the regulation of ion influx in these bacteria. The infiltration of a Xcc strain that expresses XacPNP fused to GFP in citrus leaves revealed that this protein is secreted as predicted by bioinformatics and localizes on the citrus cells plasma membrane. These results suggest that XacPNP is secreted to exert its function and that it has an effect on bacterial physiology in saline environmental conditions.

64

Session III: Pathogenesis and Regulation

P8-III

First Approach to the Discovery of Small Non-Coding RNAs in Xanthomonas axonopodis pv. manihotis

Vivian Bernal-Galeano; Andrés Rodríguez; Laura Perlaza; Adriana Bernal.

Department of Biological Sciences, Universidad de los Andes, Cr. 1 No. 18a12, Bogotá D.C., Colombia Recently, different Small noncoding RNAs (sRNA) have been discovered as relevant molecules in the regulation of several processes inside the cell. In bacteria, sRNAs are involved in post-transcriptional and post-translational regulation of quorum sensing, stress responses, virulence and infection, among others. In the genus Xanthomonas, some of them have been reported. In in this study we aimed to identify sRNAs potentially related to regulation of virulence and quorum sensing process in Xanthomonas axonopodis pv. manihotis (Xam), the causative agent of bacterial blight in cassava. We performed a transcriptomic analysis based on total RNA sequencing (library